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Uncovering synapse-specific roles of proteins implicated in complex brain disorders via novel and targeted approaches

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Uncovering synapse-specific roles of proteins implicated in complex brain disorders via novel and targeted approaches

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UNCOVERING SYNAPSE-SPECIFIC ROLES OF PROTEINS IMPLICATED IN COMPLEX
BRAIN DISORDERS VIA NOVEL AND TARGETED APPROACHES

by

Yuni Kay

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

May 2022

Copyright 2022 Yuni Kay
ii
Dedication

For 엄마, 아빠, and Ross
who made this possible through their unconditional love and support.

iii
ACKNOWLEDGEMENTS

The work in this dissertation would not have been possible without my graduate advisor,
Dr. Bruce Herring. Thank you for being generous with your time, for your endless encouragement,
and for your unrelenting belief in me. You have been an extraordinary advisor and mentor, and I
am grateful for your guidance and support over the years. I would also like to extend my sincere
gratitude to my dissertation committee members Dr. Dion Dickman, Dr. Don Arnold, and Dr.
Vsevolod Katrtich for their immense knowledge and insightful suggestions over the course of my
PhD studies.
I thank the members of the Herring lab, who have made this long journey full of excitement.
It has been an honor to work with such kindhearted and intelligent people, and I could not have
asked for better lab mates. I also thank the wider Neuroscience Graduate Program community, in
particular Dawn, Deanna, and Morgan. Many thanks to Jessica Alarcon for her helpful assistance
and for her time. I am deeply indebted to David Hong, who has been an essential figure in
facilitating scientific research across the USC campus. Thank you for inspiring me with your
incredible stories of resilience, for your kindness, for your continuous encouragement, and for your
friendship. Special thanks to my NGP friends Brendan, Artemis, Clio, and Adam Mezher, whom
I miss dearly. Thank you for our countless study sessions and for all the late nights in the library
filled with anxiety and snacks. You made the first two years of graduate school so memorable and
enjoyable, and I will always remember those years fondly because of you.
I am extremely grateful to the teachers and mentors who have left a significant impact on
me. To Kathryn and Brian – thank you for inspiring me and for teaching me the skills and
knowledge that a PhD program doesn’t. To Julie Hong, Bill Raabe, and Professor Jonathan King
iv
of Pomona College – thank you all for believing in me. I especially want to thank Professor King
for his continued mentorship and friendship over the years.
To Anna, Chen, Chiara, Lauren, and Yuna, thank you for being such fantastic friends.
Special thanks to Anna for her brilliant mind, for providing emotional support, and for being such
delightful company every day. And I must extend my deepest gratitude to Chen, who was there
for me from the beginning. Thank you for generously sharing your extensive knowledge and
ingenious suggestions - I loved learning from you. Anna and Chen, both of you have made
invaluable contributions to this work. It was such a joy to work with you, and I am privileged to
be leaving not only with a degree but also with irreplaceable friends in you.
I would like to thank my extended family all over the world. Special thanks to my uncle
and aunt who have been there for me at all the events, big and small, throughout my life. I am also
grateful to English mum, dad, and Tom for their encouragement. I give my warmest thanks to my
brother Tay and my sister Sue, both of whom I look up to and admire. Thank you for your love
and support. And a big thank you to Byul for the unending supply of joy.
Finally, none of this would have been possible without my parents and Ross. To my parents,
엄마 and 아빠, thank you for making this possible through your sacrifices and hard work. Your
profound belief in me has made me who I am today. Thank you for your unconditional love and
support. I am proud to be your daughter. I hope I made you proud.
To Ross, words cannot begin to describe how grateful I am for you. You have believed in
me through it all, and it has truly meant the world to me. How lucky I am to have you as my
practice audience, first reader, editor, and biggest supporter. Thank you most of all.

v
TABLE OF CONTENTS
Dedication ....................................................................................................................................... ii
Acknowledgements ....................................................................................................................... iii
List of Figures ............................................................................................................................... vii
Abbreviations ............................................................................................................................... viii
Abstract ........................................................................................................................................ ... x
Chapter 1: Introduction .................................................................................................................... 1
1.1 Synapses and Brain Disorders ....................................................................................... 1
1.2 Schizophrenia and Synaptic Transmission .................................................................... 5
1.3 Schizophrenia and Genetics ........................................................................................... 7
1.4 SAP97 – The Enigmatic MAGUK ................................................................................ 9
1.5 Presynaptic Dysfunction in Brain Disorders ............................................................... 12
1.6 Existing Methods of Presynaptic Investigation ........................................................... 15
Chapter 2: Schizophrenia-Associated SAP97 Mutations Increase Glutamatergic Synapse
Strength in the Dentate Gyrus and Impair Contextual Episodic Memory in Rats ........ 20
2.1 Abstract ........................................................................................................................ 20
2.2 Introduction ................................................................................................................. 21
2.3 Results ......................................................................................................................... 25
bSAP97 knockdown augments synaptic AMPAR-mediated neurotransmission
in DG granule neurons ..................................................................................... 25
bSAP97 knockdown has no effect on glutamatergic neurotransmission in CA1
pyramidal neurons ........................................................................................... 29
PSD-95, PSD-93, SAP102 knockdown similarly affects CA1 pyramidal
neurons and DG granule neurons .................................................................... 30
Inhibition of bSAP97 function in the dentate gyrus disrupts contextual episodic
memory ............................................................................................................ 31
vi
Schizophrenia-related mutations in bSAP97 release GluA1-containing
AMPARs into perforant pathway synapses ..................................................... 33
2.4 Discussion .................................................................................................................... 38
2.5 Methods ....................................................................................................................... 45
Chapter 3: An Optogenetic Method for Investigating Presynaptic Molecular Regulation ........... 84
3.1 Abstract ........................................................................................................................ 84
3.2 Introduction ................................................................................................................. 85
3.3 Results ......................................................................................................................... 88
Method Setup ............................................................................................................... 88
Method Validation 1 – Increased NMDAR-oEPSC/Fiber Volley amplitude
ratio mirrors glutamatergic synaptogenesis ..................................................... 89
Method Validation 2 – Synaptotagmin 1 Knockdown ................................................ 91
3.4 Discussion .................................................................................................................... 93
3.5 Materials and Methods ................................................................................................ 97
Chapter 4: Conclusion ................................................................................................................. 109
References ................................................................................................................................... 114

vii
LIST OF FIGURES

Figure 2.1: bSAP97 knockdown augments AMPAR-mediated neurotransmission in DG
granule neurons ............................................................................................................ 60
Figure 2.2: bSAP97 knockdown has no effect on glutamatergic neurotransmission in
CA1 pyramidal neurons. .............................................................................................. 63
Figure 2.3: PSD-95, PSD-93, SAP102 knockdown similarly affects CA1 pyramidal
neurons and DG granule neurons. ............................................................................... 65
Figure 2.4: Knockdown of bSAP97 expression in the dentate gyrus disrupts contextual
episodic memory. ......................................................................................................... 67
Figure 2.5: Clustered schizophrenia-related missense mutations in bSAP97’s PDZ2
domain release GluA1-containing AMPARs into perforant pathway synapses. ......... 69
Supplementary Figure 2.1: bSAP97 immunolabeling in the hippocampus is specific and
overlaps with MAP2 in DG granule neurons. ............................................................. 72
Supplementary Figure 2.2: Supporting data for Figure 2.1 ........................................................... 74
Supplementary Figure 2.3: Supporting data for Figure 2.2 ........................................................... 76
Supplementary Figure 2.4: Stereotaxic injection of the AAV-bSAP97-miR into the
dentate gyrus or CA1 of rats produced highly localized transduction within
each region. .................................................................................................................. 77
Supplementary Figure 2.5: Schizophrenia-related mutations in SAP97’s PDZ2 domain
are predicted to impact binding to GluA1’s PDZ-binding domain. ............................ 79
Supplementary Figure 2.6: Supporting data for Figure 2.5 ........................................................... 81
Figure 3.1: Method setup ............................................................................................................. 101
Figure 3.2: Increased NMDAR-oEPSC/Fiber Volley amplitude ratio mirrors
glutamatergic synaptogenesis .................................................................................... 103
Figure 3.3: Deficits in CA3-CA1 excitatory synaptic transmission following Syt1
knockdown in CA3 pyramidal neurons are not observed with electrical
stimulation ................................................................................................................. 105
Figure 3.4: Syt1 knockdown diminishes CA3-CA1 excitatory synaptic transmission with
optical stimulation of CA3 pyramidal neurons .......................................................... 107

viii
ABBREVIATIONS

AAV, adeno-associated virus
aCSF, artificial cerebrospinal fluid
AMPA, a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid
ASD, autism spectrum disorder
ChR2, channelrhodopsin-2
CNS, central nervous system
CNV, copy number variation
Co-IP, co-immunoprecipitation
DG neurons, dentate granule neurons
DG, dentate gyrus
DIV, days in vitro
eEPSC, evoked/electrically evoked excitatory postsynaptic currents
eIPSC, evoked inhibitory postsynaptic currents
FV, fiber volley
GEF1, guanine nucleotide exchange factor 1
GWAS, genome-wide association study
KD, knockdown
MAGUK, membrane-associated guanylate kinase
miR, microRNA
NMDA, N-methyl-D-aspartate
oEPSC, optically induced excitatory postsynaptic currents
PDZ, PSD-95/Discs large/Zona occludens-1
ix
PNS, peripheral nervous system
PSD, postsynaptic density
P_, postnatal day _
RNAi, RNA interference
SEM, standard error of the mean
SH3, Src-homology-3
Syt1, synaptotagmin 1

x
ABSTRACT

Dysfunction of glutamatergic neurotransmission underlies the pathophysiology of various
complex brain disorders, such as schizophrenia. While the exact etiology of schizophrenia remains
to be elucidated, accumulating evidence has implicated the MAGUK protein SAP97 in
schizophrenia. However, SAP97’s relevance in glutamatergic neurotransmission has long
remained uncertain. In Chapter 2, we employ a targeted approach to uncover SAP97’s synaptic
role by first visually identifying the dentate gyrus, a hippocampal region independently implicated
in schizophrenia, as where endogenous SAP97 may play a synaptic regulatory role. We find that
schizophrenia-related perturbations of SAP97 produce significant augmentation of glutamatergic
neurotransmission in the dentate gyrus but not in the CA1 region, where SAP97 was previously
studied despite little endogenous expression in this region. Further, disrupting SAP97 function in
this region impairs contextual episodic memory in rats, altogether demonstrating that the synapse
regulatory mechanism involving SAP97 in this region may contribute to the development of
symptoms associated with schizophrenia. Chapter 3 highlights the significance of the other half of
the synapse in regulating glutamatergic neurotransmission and maintaining healthy brain function,
as mutations in key presynaptic proteins have also been strongly implicated in complex brain
disorders. We introduce a novel method designed to expedite our understanding of molecular
regulatory pathways that govern presynaptic function, and we utilize this method to determine
synaptotagmin 1’s presynaptic role in the Schaffer collateral-CA1 synapse. This new method may
now be leveraged to bolster our understanding of the presynaptic roles of other proteins implicated
in complex brain disorders. Altogether, this dissertation uncovers the synapse-specific regulatory
roles of proteins implicated in complex brain disorders, and my hope is that this work will
contribute to the development of new strategies to more effectively treat these disorders.
1
CHAPTER 1: INTRODUCTION

1.1 Synapses and Brain Disorders

Although the brain remains the most mysterious and complex organ in the human body, it
is understood that synaptic transmission underlies the basic function of the brain. Synaptic
transmission in the vertebrate central nervous system refers to the communication between two
neurons where signals are transmitted from one neuron to the other through the small space
between them called a synapse. The critical role of synaptic transmission in brain function was
made evident by the findings that even small disruptions in synaptic transmission can lead to
neurological disorders (Lepeta et al., 2016, Zoghbi and Bear, 2012, Boda et al., 2010, Bourgeron,
2015, Grant, 2012). In synaptic transmission, the neuron that is sending the signal and initiating
the communication is called the presynaptic neuron, and the neuron that is receiving the
information is called the postsynaptic neuron. The presynaptic neuron releases vesicles containing
signaling molecules called neurotransmitters into the synaptic junction, and the postsynaptic
neuron receives these neurotransmitters via receptors. The resulting postsynaptic response is
specific to both the type of neurotransmitter released by the presynaptic neuron and the type of
receptors expressed on the postsynaptic neuron (Fröhlich, 2016).
There are many types of neurotransmitters, which can be functionally categorized as
modulatory, inhibitory, or excitatory. Modulatory neurotransmitters regulate the function of
groups of neurons, inhibitory neurotransmitters dampen neuronal activity, and excitatory
neurotransmitters increase activity. Dopamine is a modulatory neurotransmitter that has both
inhibitory and excitatory effects depending on the receptors (Akaike et al., 1987, Kandel et al.,
2013). It plays a critical modulatory role in several systems, including reward and reinforcement,
2
regulation of hormones, as well as movement and motor control (Klein et al., 2019). Dopamine is
implicated in mood disorders (Grace, 2016, Delgado, 2000) and neurodevelopmental disorders
(Cai et al., 2021, Paval, 2017), but dopaminergic dysfunction is most famously responsible for
Parkinson’s disease (Davie, 2008, Fahn, 2008). The main inhibitory neurotransmitter in the brain
is GABA. Problems with GABAergic transmission can also lead to disorders, including mood
disorders (Brambilla et al., 2003, Fogaca and Duman, 2019), epilepsy (Treiman, 2001), and
Huntington’s disease (Garret et al., 2018).
Glutamate is the major excitatory neurotransmitter in the brain. Almost all excitatory
synapses in the vertebrate central nervous system are glutamatergic, and it is estimated that more
than half of all neurons in the brain release glutamate (Purves, 2001). Glutamatergic
neurotransmission occurs via two types of receptors: metabotropic and ionotropic glutamate
receptors. Metabotropic glutamate receptors, or mGluRs, indirectly modulate postsynaptic
response by binding to G-proteins that ultimately trigger downstream signal transduction pathways
(Purves, 2001). On the other hand, ionotropic glutamate receptors are themselves ligand-gated ion
channels that open upon binding to glutamate and allow cations such as Na
+
, K
+
, and sometimes
Ca
2+
to flow. These receptors, formed by tetrameric complexes of subunits, include AMPA (a-
amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid) receptors and NMDA (N-methyl-D-
aspartate) receptors, each named after the agonists that activate them (Niciu et al., 2012). Both
AMPA and NMDA receptors have been thoroughly characterized because of the significant roles
they play in synaptic plasticity. Synaptic plasticity is activity-dependent strengthening or
weakening of synaptic transmission, a process which is believed to underly learning and memory
(Kandel et al., 2013, Meldrum, 2000, Niciu et al., 2012). AMPA and NMDA receptor mediated
signaling, and glutamatergic neurotransmission in general, have been implicated in the etiology of
3
neurodevelopmental disorders, neurodegenerative disorders, and psychiatric disorders that cause
substantial societal and economic burden (Javitt, 2004, Volk et al., 2015, Li et al., 2018, Benarroch,
2018, Rojas, 2014). In particular, the role of glutamatergic neurotransmission in autism spectrum
disorder (ASD) has been recently garnering significant interest.
Although the exact etiology of ASD remains unclear, growing evidence has linked altered
glutamatergic synapse structure and function with ASD (Volk et al., 2015, Bourgeron, 2015, Rojas,
2014). ASD is a neurodevelopmental disorder, and although it is a “spectrum” disorder due to the
considerable variation in symptoms and severity across individuals, the diagnostic criteria of ASD
include difficulty in communication and social interactions, restricted interests, and repetitive
patterns of behavior (American Psychiatric Association, 2013). Cognitive impairment is another
common characteristic of ASD, and the rate of comorbidity of ASD and intellectual disability is
estimated to be around 75% (Mpaka et al., 2016, Lai et al., 2014). The CDC’s Autism and
Developmental Disabilities Monitoring Network found that the prevalence of ASD is about one in
44 children aged 8 years (Maenner et al., 2021). While ASD does not have one single cause, it is
widely accepted that genetic abnormalities play a significant role (Lai et al., 2014), with studies
finding high heritability of ASD (Bai et al., 2019, Sandin et al., 2017, Wei et al., 2020, Tick et al.,
2016) and large exome sequencing studies identifying numerous candidate ASD risk genes
(Iossifov et al., 2014, Yuen et al., 2015, Feliciano et al., 2019, Satterstrom et al., 2020). One
promising candidate ASD risk gene TRIO, previously determined to be required for glutamatergic
neurotransmission (Herring and Nicoll, 2016), was found to harbor a cluster of ASD-related de
novo mutations in its GEF1 domain (Sadybekov et al., 2017). In-depth, functional analyses of these
mutations revealed that every identified ASD-related de novo mutation in the TRIO hotspot
significantly altered glutamatergic synapse function in the hippocampus, further implicating
4
glutamatergic synaptic dysfunction in the etiology of ASD (Sadybekov et al., 2017). Another
disorder likely involving glutamatergic dysfunction is schizophrenia, the etiology of which is
arguably even more complex and enigmatic.

5
1.2 Schizophrenia and Synaptic Transmission

Schizophrenia affects more than 24 million people worldwide (WHO, 2022). As one of the
world’s leading causes of disabilities, schizophrenia not only impacts the individuals’ well-being
but also confers significant economic and humanistic burdens (Millier et al., 2014, Chong et al.,
2016). Despite extensive research efforts over decades, no significant breakthroughs have been
made in determining the etiology of this neuropsychiatric disorder (Igolkina et al., 2018). The
prevailing theory has been centered around disruption in the dopaminergic system in the brain,
particularly through hyperactive dopaminergic synaptic transmission. This dopamine hypothesis
of schizophrenia is supported by the fact that antipsychotic medications that have successfully
treated the symptoms of schizophrenia are dopamine receptor antagonists, such as clozapine and
haloperidol (Kapur and Remington, 2001, Grace, 2016, Javitt and Zukin, 1991, Seeman et al.,
1975). These antipsychotics have been the primary method of treatment for schizophrenia over the
last 50 years, but these compounds directed at D2-type dopamine receptors have only been
successful in treating the positive symptoms of schizophrenia but not the negative symptoms or
the cognitive deficits, which are the key predictors of functional disability (Moghaddam and Javitt,
2012, Kirkpatrick et al., 2006). With no significant advancements in development of more
efficacious compounds that can relieve the core debilitating symptoms of schizophrenia, attention
has shifted to identifying better drug targets, such as the glutamatergic synapse (Moghaddam and
Javitt, 2012).
The glutamate hypothesis of schizophrenia is another leading theory of the etiology and
pathophysiology of the disorder. Glutamate disturbance was implicated in schizophrenia when
NMDAR antagonists such as phencyclidine (PCP), MK801, and ketamine led to immediate
symptoms in humans that resembled the positive, negative, and cognitive symptoms of
6
schizophrenia (Howes et al., 2015, Javitt and Zukin, 1991), and that these antagonists exacerbated
these symptoms in individuals already diagnosed with schizophrenia (Krystal et al., 2003). This
hypothesis was further substantiated by studies in both animal models and in humans, with findings
such as reduction of the NMDAR subunit GluN1 in the hippocampus of individuals with
schizophrenia (Gao et al., 2000), inhibition of NMDAR function in animal models leading to
schizophrenia-like behaviors (Jones et al., 2011), and hippocampal hypermetabolism and atrophy
in MRI imaging of individuals with psychosis, a result which was mirrored in ketamine-induced
mouse models as well as following administration of glutamate reducing drug LY379268 in mice
(Schobel et al., 2013). Further, several genes involved in glutamatergic neurotransmission were
implicated in schizophrenia through GWAS studies (Schizophrenia Working Group of the
Psychiatric Genomics, 2014). Most notably, the largest sequencing study of schizophrenia to date
by the Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) consortium identified rare
coding variants in 10 schizophrenia risk genes, 4 of which were either directly or indirectly
involved in glutamatergic neurotransmission (Singh et al., 2020): GRIN2A - encoding NMDAR
receptor subunit GluN2A, GRIA3 - encoding AMPA receptor subunit GluA3, TRIO – encoding a
Rho guanine nucleotide exchange factor that has been shown to directly affect glutamatergic
neurotransmission (Herring and Nicoll, 2016, Sadybekov et al., 2017), and SP4 – encoding Sp4
transcription factor, which has been shown to impact NMDAR-mediated neurotransmission (Zhou
et al., 2010, Priya et al., 2013). Taken together, such findings all point to glutamate dysfunction as
a compelling driver of schizophrenia.

7
1.3 Schizophrenia and Genetics

Although the non-Mendelian pattern of inheritance suggests that there is a significant
environmental influence involved, the high heritability of schizophrenia has established that
genetics likely contributes to the risk of developing schizophrenia. The genetics of schizophrenia
is complex and still not well understood. Early work in this field identified a number of candidate
risk genes, notably the Disrupted in Schizophrenia 1 gene, or DISC1. DISC1 encodes an
intracellular scaffold protein that interacts with many other proteins and is indirectly involved in
numerous facets of neuronal function including in neural development and synapse function
(Brandon and Sawa, 2011, Camargo et al., 2007). DISC1, and the possibility of genetics
contributing to psychiatric illness, came to attention when a Scottish family with a high load of
mental illness was discovered harboring a rare chromosomal translocation severing the mid-point
of the gene (St Clair et al., 1990, Millar et al., 2000, Blackwood et al., 2001). Another study
identified an American family with schizophrenia and schizoaffective disorder who harbored a
frameshift mutation in DISC1 (Sachs et al., 2005). However, further research on DISC1’s role in
schizophrenia has led to mixed results and controversy, especially after recent, high-powered
GWAS studies did not find an association between DISC1 and schizophrenia (Schizophrenia
Working Group of the Psychiatric Genomics, 2014, Sullivan, 2013, Porteous et al., 2014, Facal
and Costas, 2019, Johnstone et al., 2015, Farrell et al., 2015, Niwa et al., 2016).
Recent advancements in the field of schizophrenia genetics, made possible through
significant technological developments as well as large-scale collaborations, have led to
identification of several types of genetic variants that increase schizophrenia risk, which include
de novo variants, 100+ loci harboring common risk variants, and rare (<1%) and large (>100kb)
copy number variants (CNVs) (Avramopoulos, 2018). The first CNV explored in schizophrenia
8
relevance was the 22q11.2 deletion. Deletion of the chromosomal band 22q11.2 was found to cause
velo-cardio-facial syndrome, and intriguingly there were reports of comorbidity with psychosis
(Chow et al., 1994). Further screening following this discovery led to identification of more
individuals with this comorbidity (Lindsay et al., 1995, Karayiorgou et al., 1995). The 22q11.2
deletion results in the loss of more than 50 genes including COMT, another extensively studied
candidate risk gene from earlier schizophrenia studies. Although the 22q11.2 deletion is arguably
the most researched CNV in schizophrenia, the CNV that has been shown to confer the highest
known risk for developing schizophrenia is the 3q29 microdeletion, which confers a greater than
40-fold increase in the risk (Mulle, 2015). The exome sequencing study by the SCHEMA
consortium indicated that the 3q29 microdeletion has the highest odds ratio and the lowest minor
allele frequency in the general population of all known genetic perturbations that have reached
whole genome significance for schizophrenia (Singh et al., 2020). The 3q29 microdeletion
syndrome is a rare genetic disorder that occurs in about one in 30,000 individuals (Glassford et al.,
2016). It is characterized by a variety of neurodevelopmental and neuropsychiatric phenotypes,
including intellectual disability, motor weakness, anxiety disorders, ADHD, and psychosis
(Sanchez Russo et al., 2021). In the 3q29 microdeletion syndrome, 22 genes are deleted, of which
DLG1 is considered to be one of the most promising candidate genes for a variety of reasons,
starting with the fact that schizophrenia is believed to be a synaptic disorder and that the protein
that DLG1 gene encodes, SAP97, belongs to a family of proteins that have been strongly
implicated in brain disorders.

9
1.4 SAP97 – The Enigmatic MAGUK

SAP97 is a member of the membrane-associated guanylate kinase homologs, or MAGUK
protein family. The MAGUK protein family plays an essential role in the assembly, function, and
regulation of the scaffolding network in glutamatergic synapses (Won et al., 2017). MAGUKs
have been further divided into subfamilies based on genomic sequences of their core PDZ-SH3-
GUK region as well as their unique, additional protein domains. Of these, proteins of the discs
large (DLG) subfamily consist of SAP97, PSD-95, PSD-93, and SAP102. These MAGUKs
contain three PSD-95/Discs large/Zona occludens-1 (PDZ) domains, a Src-homology-3 (SH3)
domain, and a guanylate kinase (GUK) domain that is catalytically inactive due to the lack of a P-
Loop that binds ATP (Oliva et al., 2012). The roles of PSD-95, PSD-93, and SAP102 in the brain
have already been well-characterized (Won et al., 2017). However, despite its structural similarity
to the other MAGUKs, the role of SAP97 in neurons has remained elusive.
While MAGUKs share a common domain organization, they differ significantly in the
sequences between these domains (Montgomery et al., 2004). SAP97, PSD-95, and PSD-93
contain alternative N-termini expressing either an L27 domain, leading to b isoforms of each, or
a palmitoylation motif of double cysteines, leading to a isoforms. PSD-95 and PSD-93 are mainly
expressed in their a isoforms, but SAP97 is primarily expressed in its b isoform in the brain (Fourie
et al., 2014). Alternative splicing of the N-terminus allows for specific localization of each isoform
of SAP97, targeting a-SAP97 to the postsynaptic density (PSD) and targeting b-SAP97 to
perisynaptic regions of glutamatergic synapses (Waites et al., 2009).
Also in the N-terminus of SAP97 before the first PDZ domain are two spliced insertions,
I1A and I1B. Isoforms containing only the I1A insert but not I1B were not detected in any human
tissue, and I1A was found to be barely detectable in the human brain (McLaughlin et al., 2002,
10
Mori et al., 1998). The region between the SH3 and GUK domains of SAP97 contains four spliced
insertions: I2, I3, I5, and I4. Isoforms containing six combinations of these insertions exist in the
brain. Of those, I3I5, I2I5I4, and I2I5 are most abundant in the brain (McLaughlin et al., 2002).
The existence of such a large variety of isoforms may be one of the reasons why SAP97 is
considerably less understood compared to the other MAGUKs. This is compounded by the fact
that older SAP97 studies often did not identify which isoform they were characterizing (Fourie et
al., 2014). Experiments discussed in Chapter 2 employ the bSAP97-I1BI3I5 isoform, as it is the
best characterized and most relevant isoform in synaptic function (Nikandrova et al., 2010,
Schluter et al., 2006, Rumbaugh et al., 2003, Waites et al., 2009).
Another potential reason why SAP97’s role has remained inconclusive is that its expression
pattern differs from the other MAGUKs. In the hippocampus, the Allen Brain Atlas shows that
while DLG2 (PSD-93), DLG3 (SAP102), and DLG4 (PSD-95) mRNA are expressed mostly
evenly across CA3 pyramidal neurons, CA1 pyramidal neurons, and dentate granule neurons (DG
neurons), DLG1 mRNA is concentrated in the DG neurons in comparison to the CA3 and CA1
pyramidal neurons. This difference in gene expression pattern may be another reason why
SAP97’s synaptic function has been inconsistent in literature, based on assumptions that most
glutamatergic synapses are similar enough to draw overarching conclusions based on studying a
particular synapse, such as the Schaffer collateral-CA1 synapse. Chapter 2 will discuss the pitfalls
of such a general approach and the discovery of SAP97’s function where it is endogenously
expressed, in the dentate gyrus of the hippocampus.
As would be expected from the critical synaptic roles of MAGUKs, mutations in MAGUKs
have been shown to have strong implications in neurological disorders. PSD-95 has been
implicated in ASD, and SAP102 has been implicated in intellectual disability (Zanni et al., 2010,
11
Crocker-Buque et al., 2016, Tarpey et al., 2004, Coley and Gao, 2018). PSD-95, PSD-93, and
SAP97 have all been implicated in schizophrenia (Xing et al., 2016). Of these, SAP97 has the
strongest connection to schizophrenia, because DLG1 is one of the genes deleted in the 3q29
microdeletion which confers the largest risk of developing schizophrenia of any known genetic
perturbation to date (Mulle, 2015). Additionally, three individuals with schizophrenia were
identified with missense mutations clustered in the PDZ2 domain of SAP97, and two of these
unrelated individuals were found to harbor the exact identical missense mutation (Fromer et al.,
2014, Xing et al., 2016). While it is likely that these mutations contribute to the development of
schizophrenia in these individuals, it has been difficult to understand this relationship because of
the lack of knowledge on the role of SAP97 in the brain. SAP97’s newly discovered synaptic role
and its potential schizophrenia relevance will be discussed in Chapter 2.

12
1.5 Presynaptic Dysfunction in Brain Disorders

Much of the discussion on the role of synaptic proteins in brain disorders has focused on
postsynaptic disturbances thus far, but unsurprisingly, healthy brain function also relies on normal
presynaptic function (Waites and Garner, 2011, Jahn and Fasshauer, 2012). Mutations in key
presynaptic proteins involved in regulation of neurotransmitter release such as synapsins,
syntaxins, and SNAP25 have been implicated in shared neurodevelopmental phenotypes, such as
movement disorders, epilepsies, intellectual disabilities, ASD, and psychosis (John et al., 2021,
Fassio et al., 2011, Guarnieri et al., 2017, Cartier et al., 2015, Schubert et al., 2014, Heyne et al.,
2018, Rohena et al., 2013). Along with trafficking, docking, and priming of synaptic vesicles for
release, a fundamental step in presynaptic neurotransmitter release is the precise temporal
coordination of vesicle fusion. This temporal property of vesicle release is regulated by a family
of proteins called the synaptotagmins (Sudhof, 2002, Sudhof, 2014). Synaptotagmins regulate the
speed and coordination of vesicular exocytosis by serving as the calcium sensor following action
potential-induced calcium influx in the presynaptic terminal. Although all synaptotagmins contain
two C-terminal C2 domains, C2A and C2B, only eight of 17 synaptotagmins contain the C2
domains that impart the calcium sensitivity which allows them to trigger exocytosis (MacDougall
et al., 2018). These eight synaptotagmins have been found to exhibit different levels of calcium
sensitivity, and these differences in calcium sensitivities are believed to be responsible for the
distinct temporal dynamics of each of the synaptotagmins. For example, the high-affinity, slow-
binding synaptotagmins such as Syt7 are often found localized to plasma membranes and are
believed to function as sensors for slow calcium-dependent exocytosis, whereas the low-affinity,
fast-binding synaptotagmins such as Syt1 and Syt2 are localized to synaptic vesicles (Sugita et al.,
2002). Syt1 and Syt2 are believed to be the major calcium sensors responsible for regulation of
13
fast and synchronous neurotransmitter release in the brain (Geppert et al., 1994, Maximov and
Sudhof, 2005, Xu et al., 2007, Bacaj et al., 2013). And of all the synaptotagmins, Syt1 is the most
studied and best characterized isoform.
Syt1’s critical role in brain function was recently brought to the spotlight after the
discovery of the Syt1-associated neurodevelopmental disorder in 11 patients (Baker et al., 2018,
Baker et al., 2015). Also called the Baker-Gordon Syndrome, the disorder is characterized by
infantile hypotonia, ophthalmic abnormalities, moderate to profound global developmental delay,
poor or absent speech, behavioral abnormalities, hyperkinetic movements, and EEG abnormalities
in the absence of overt seizures (Baker et al., 2018). Notably, all 11 identified patients had de novo
heterozygous missense mutations in Syt1 clustered in the C2B domain. Further, the magnitude of
the disruption in synaptic kinetics caused by these clustered de novo mutations paralleled the
severity of the neurodevelopmental disorder phenotype in the patients. Such clustering of
mutations within a particular domain, as was found in the GEF1 domain of Trio in individuals with
ASD or ASD-related disorders (Sadybekov et al., 2017) and in the PDZ2 domain of SAP97 in
individuals with schizophrenia (Fromer et al., 2014, Xing et al., 2016) (will cite mine), helps lead
to the identification of the mechanisms behind the vital synaptic roles of these proteins. The Syt1-
associated disorder provides yet another example of how dysregulation of synapses can give rise
to neurodevelopmental disorders, but the other proteins described so far such as Trio and SAP97
are largely considered postsynaptic proteins and were thus investigated from the postsynaptic side,
whereas Syt1 has a well-established presynaptic role. Even though studying each side of the
synapse is imperative in studying synaptic transmission, especially given that many presynaptic
proteins have been implicated in development of neurodevelopmental disorders (John et al., 2021),
14
the availability of optimized methods for studying the presynaptic side of glutamatergic synapses
has been lacking compared to the postsynaptic side.

15
1.6 Existing Methods of Presynaptic Investigation

Refined and efficient methods already exist for studying the impact of genetic manipulation
of the postsynaptic side of glutamatergic synapses. One such method employed in both Chapters
2 and 3 uses biolistic transfection to target the genetic manipulation to postsynaptic neurons in rat
organotypic hippocampal slice cultures (Sadybekov et al., 2017, Elias et al., 2008, Schnell et al.,
2002, Tian et al., 2018, Paskus et al., 2019, Rao et al., 2019, Herring et al., 2013, Herring and
Nicoll, 2016). On the presynaptic side, there are fewer commonly utilized preparations that have
been instrumental in our understanding of the cellular and molecular biology of synaptic vesicle-
mediated neurotransmitter release. While these methods each have unique advantages, they also
have notable limitations.
One preparation for studying the cellular and molecular biology of presynaptic vesicle-
mediated neurotransmitter release utilizes the Drosophila larval neuromuscular junction (NMJ).
The NMJ exhibits both developmental and functional plasticity, and it has been studied extensively
to reveal its similarities to synapses of vertebrate nervous systems (Menon et al., 2013). NMJ
synaptic boutons are large and easily accessible, allowing for both imaging and
electrophysiological recordings. A single presynaptic axon can be electrically stimulated in order
to carefully characterize the presynaptic release machinery (Kopke and Broadie, 2018).
Drosophila are also genetically malleable, and the simplicity of the organism allows for forward
genetic screenings (Bykhovskaia and Vasin, 2017). The combination of these unique advantages
led to the Drosophila larval NMJ being one of the most utilized models for studying the
glutamatergic synapse. The main disadvantage with this setup is that Drosophila are not mammals.
Furthermore, the NMJ is not a neuron-to-neuron, central nervous system (CNS) synapse, but rather
a neuron-to-muscle connection in the peripheral nervous system (PNS), making the process of
16
generalizing the results from the Drosophila NMJ to mammalian CNS glutamatergic synapses a
bit more complex.
A mammalian model system that has allowed for extensive studies in presynaptic vesicle
release employs adrenal chromaffin cells, which are neuroendocrine cells of the adrenal medulla.
These cells, commonly isolated from cows, mice, and rats, allow for direct measurement of
released catecholamines via carbon fiber amperometry, as well as simultaneous measurement of
electrical membrane capacitance via voltage-clamp (Neher, 2006). Using chromaffin cells as a
model system has led to many discoveries regarding the processes of docking, priming, and
releasing of vesicles (Neher, 2018). While the two independent and simultaneous measurements
of secretion makes the adrenal chromaffin cells an attractive presynaptic model system, there are
significant differences between the processes of catecholamine release by chromaffin cells and
neurotransmitter release by neurons, which include: 1) active zones are not readily visible in
chromaffin cells, 2) the release sites are not tightly coupled to sites of calcium entry, 3) there is no
rapid recycling of the released vesicles in chromaffin cells, 4) chromaffin cells only release
catecholamines, and 5) chromaffin cells are not neurons (Stevens et al., 2011). These reasons make
it difficult to draw generalizable conclusions from endocrine vesicle release to neuronal
neurotransmitter release.
The calyx of Held, another prevalent model system, addresses some of these issues and
allows for study of a glutamatergic synapse of the mammalian central nervous system. The calyx
of Held is a synapse in the auditory brainstem, and it is described to be the largest synapse in the
brain (Morest, 1968). The large size of this synapse leads to technically easier patch-clamp
recordings. But more importantly, the size confers the distinctive advantage of simultaneous pre-
and postsynaptic voltage clamp (Neher, 2006). Because the postsynaptic neuron receives input
17
from a single presynaptic neuron, with simultaneous voltage-clamp, deconvolution analysis can
be used with the recorded size and shape of the currents to determine the amount of transmitter
release as well as the kinetics and density of postsynaptic receptors (Wang et al., 2009). The
accessibility of the synapse also permits simultaneous electrophysiological and calcium-imaging
studies (Baydyuk et al., 2016). Studies in this model synapse have led to significant discoveries in
the machinery behind both endocytosis and exocytosis, as well as in ion channels and short-term
plasticity. However, the calyx of Held is not representative of a typical glutamatergic synapse in
the mammalian CNS because of its extraordinarily large size and the fact that the calyx of Held
contains several hundred active zones. Additionally, the calyx of Held is an axosomatic synapse,
which may account for the notably fast and efficient synaptic transmission (Borst and Soria van
Hoeve, 2012).
One of the most common methods for studying typical synapses of the mammalian CNS
uses dissociated neuronal cultures. For studying glutamatergic synapses, neurons are most often
cultured from the hippocampus. The main advantage of this setup is that it is relatively easy to
make presynaptic genetic manipulations in these cultures to assess how the changes in presynaptic
vesicle release influences the resulting postsynaptic current via voltage-clamping. Cultured
neurons can also be prepared from transgenic mice. However, dissociating the neurons prior to
culturing means that the specific connections between neuronal subtypes are lost. In the culture
dish, dissociated neurons form synapses with other neurons regardless of subtype, meaning these
connections are not necessarily representative of naturally occurring synapses. Furthermore, it is
becoming increasingly more evident that neurons of different subregions of the hippocampus have
different properties (Rao et al., 2019, Siddiqui et al., 2013, Farris et al., 2019, Alkadhi, 2019, Zhu
et al., 2018). Therefore, it may not be appropriate to make generalizations about all hippocampal
18
synapses based on results from synapses between unidentified subtypes of hippocampal neurons
in a dish. Another concern is that in some studies of presynaptic proteins, the genetic manipulation
is made in all of the neurons, not just the presynaptic neuron. Often, the manipulation of a particular
protein in the presynaptic neuron is interpreted as the reason for observed postsynaptic changes.
This, however, may not be true, as the “presynaptic” protein of focus may also have an unknown
postsynaptic role. To accurately determine the presynaptic role of a protein, the genetic
manipulation must be limited to the presynaptic neuron. This is possible through sparse
transfections with fluorescent markers, but often this distinction is not made.
Finally, the current gold standard of methods for studying presynaptic vesicle release in
typical, naturally occurring synapses of mammalian CNS neurons is using transgenic animals.
With transgenic mice, both in vivo and in vitro studies in specific synapses of interest is possible,
and unlike with the dissociated neurons, the brain’s endogenous circuitry remains intact. The major
problem with this model system is practicality: transgenic mice are expensive and difficult to
generate in a timely manner. In order to study multiple proteins, or even the same protein with
mutations or different isoforms, each would require a newly generated transgenic mouse line.
Another drawback is that some genetic manipulations may be developmentally lethal, meaning
they cannot be studied using transgenic animals. Recognizing these limitations of the existing
model systems for presynaptic vesicle release, we have developed a new and efficient optogenetic
technique to genetically manipulate and study presynaptic neurons at naturally occurring synapses
in an intact neuronal circuit in the mammalian CNS. This novel method will be discussed in
Chapter 3.
This dissertation will discuss the discovery of the synapse-specific roles of two proteins,
SAP97 and Syt1, both of which have been implicated in complex brain disorders. Chapter 2 will
19
discuss the perforant path-dentate gyrus synapse regulatory mechanism involving SAP97 that,
when disrupted, may contribute to the development of symptoms associated with schizophrenia.
Chapter 3 will discuss a novel optical/electrophysiological method designed to expedite our
understanding of molecular regulatory pathways that govern presynaptic function, and the
utilization of this method to uncover Syt1’s presynaptic role in the Schaffer collateral-CA1 synapse.
This new method may now be applied to bolster our understanding of the presynaptic roles of other
proteins implicated in complex brain disorders. Altogether, the work described in this dissertation
stands to aid in the development of new strategies to more effectively treat such disorders.
20
CHAPTER 2: SCHIZOPHRENIA-ASSOCIATED SAP97 MUTATIONS
INCREASE GLUTAMATERGIC SYNAPSE STRENGTH IN THE
DENTATE GYRUS AND IMPAIR CONTEXTUAL EPISODIC MEMORY
IN RATS

2.1 Abstract

Mutations in the putative glutamatergic synapse scaffolding protein SAP97 are associated with the
development of schizophrenia in humans. However, the role of SAP97 in synaptic regulation is
unclear. Here we show that SAP97 is expressed in the dendrites of granule neurons in the dentate
gyrus but not in the dendrites of other hippocampal neurons. Schizophrenia-related perturbations
of SAP97 did not affect CA1 pyramidal neuron synapse function. Conversely, these perturbations
produce dramatic augmentation of glutamatergic neurotransmission in granule neurons that can be
attributed to a release of perisynaptic GluA1-containing AMPA receptors into the postsynaptic
densities of perforant pathway synapses. Furthermore, inhibiting SAP97 function in the dentate
gyrus was sufficient to impair contextual episodic memory. Together, our results identify a cell-
type-specific synaptic regulatory mechanism in the dentate gyrus that, when disrupted, impairs
contextual information processing in rats.

21
2.2 Introduction

Schizophrenia is a debilitating psychiatric disorder that affects around 20 million people
worldwide. Symptoms of this disorder include hallucinations, delusions, flat affect, the loss of a
sense of personal identity, poor executive function, and deficits in memory (Yun et al., 2016, Kahn
et al., 2015). Despite many years of research, it remains unclear which cellular and molecular
mechanisms in the brain are disrupted in individuals with schizophrenia. It is also unclear which
specific regions in the brain are strongly affected by the disruption of these mechanisms.
Accumulating evidence supports a connection between SAP97 loss of function and schizophrenia
(Toyooka et al., 2002, Kushima et al., 2018, Soler et al., 2018, Carroll et al., 2011, Purcell et al.,
2014, Mulle et al., 2010, Mulle, 2015, Sato et al., 2008, Uezato et al., 2012, Xing et al., 2016,
Marshall et al., 2017, Fromer et al., 2014). For example: the gene encoding SAP97, DLG1, was
recently identified as a potential hub of schizophrenia-related synaptic dysfunction (Marshall et
al., 2017); missense mutations in SAP97 have been identified in individuals with schizophrenia
(Fromer et al., 2014, Xing et al., 2016); and microdeletion mutations in humans resulting in the
loss of a DLG1 allele give rise to a 40-fold increase in the risk of developing schizophrenia (Mulle
et al., 2010, Mulle, 2015). SAP97 is a Membrane-Associated Guanylate Kinase (MAGUK) protein.
MAGUK protein family members include PSD-95, PSD-93, SAP102, and SAP97. PSD-95, PSD-
93, and SAP102 are major constituents of the glutamatergic synapse postsynaptic density (PSD).
At the postsynaptic side of glutamatergic synapses, PSD-95, PSD-93, and SAP102 provide
scaffolds for synaptic protein complexes via their three PSD-95/Dlg/ZO1 (PDZ) domains (Won et
al., 2017). It is well established that PSD-95, PSD-93, and SAP102 use their PDZ domains to
anchor AMPA and NMDA receptors (AMPARs and NMDARs) in the PSD in direct opposition to
regions of presynaptic glutamate release. This MAGUK protein-mediated glutamate receptor
22
positioning is critical for efficient activation of these receptors by presynaptically-released
glutamate.
In contrast to other MAGUK proteins, the role of SAP97 in synaptic regulation is uncertain,
despite the fact that SAP97 is the only MAGUK that can interact directly with AMPARs. This
interaction occurs through SAP97’s PDZ2 domain binding to the C-terminal PDZ-binding domain
of the AMPAR subunit GluA1 (Leonard et al., 1998, Sans et al., 2001, Kim et al., 2005, Zhou et
al., 2008, Cai et al., 2002). It is also unclear whether the C-tail of GluA1 plays any role in synaptic
regulation (Granger et al., 2013, Kim et al., 2005). Previous studies have provided conflicting data
regarding SAP97’s influence on synaptic function (Nakagawa et al., 2004, Schnell et al., 2002,
Schluter et al., 2006, Li et al., 2011, Ehrlich et al., 2007, Rumbaugh et al., 2003). Most of these
studies have relied on recombinant expression of SAP97 and/or dissociated neuron cultures where
synapses form between unknown neuron subtypes. However, one particularly notable study has
shown that knocking out SAP97 led to no alteration in glutamatergic neurotransmission in
hippocampal CA1 pyramidal neurons (Howard et al., 2010). Additionally, mutant mice expressing
a PDZ-binding domain lacking form of the GluA1 subunit which prevents AMPARs from binding
to SAP97 (GluA1-D7 mice) were found to have normal glutamatergic neurotransmission in
hippocampal CA1 pyramidal neurons (Kim et al., 2005). Together, these studies have been used
to support a compelling argument against a role for SAP97 in the regulation of glutamatergic
synaptic transmission (Won et al., 2017). This lack of understanding regarding SAP97’s role in
the brain has been particularly frustrating given SAP97’s growing implication in schizophrenia,
SAP97’s similarity to other essential synaptic proteins, and the fact that schizophrenia is largely
considered to be a synaptic disease (Marshall et al., 2017, Fromer et al., 2014, Volk et al., 2015).
Increasing evidence points to dysfunction of the dentate gyrus as a contributing factor in
23
the development of schizophrenia (Yun et al., 2016, Das et al., 2014, Tamminga et al., 2010,
Kawano et al., 2015, Ota et al., 2017, Falkai et al., 2016, Kirov et al., 2013, Jaffe et al., 2020,
Nakahara et al., 2019, Tavitian et al., 2019). The dentate gyrus serves as the gateway for
information coming into the hippocampus via the perforant pathway, and symptoms associated
with schizophrenia exhibit a high degree of correlation with symptoms stemming from dentate
gyral dysfunction (Berron et al., 2016, Yun et al., 2016, Das et al., 2014). In the present study we
find that expression of bSAP97, the major isoform of SAP97 (Schluter et al., 2006), is absent in
the apical and basal dendrites of CA3 and CA1 pyramidal neurons of the hippocampus. In contrast,
robust bSAP97 expression is observed in the dendrites of granule neurons in the dentate gyrus
(DG granule neurons). Given that reduced SAP97 expression in the brain is associated with a
substantially increased risk of developing schizophrenia (Mulle et al., 2010, Mulle, 2015, Toyooka
et al., 2002), we were interested in whether bSAP97 regulates glutamatergic synapse function in
DG granule neurons and whether reduced bSAP97 expression in the dentate gyrus is sufficient to
produce schizophrenia-related behavioral phenotypes in rodents.
Here we find that reducing bSAP97 expression in DG granule neurons results in a dramatic
increase in AMPAR-mediated synaptic transmission following perforant path stimulation.
Conversely, inhibition of bSAP97 expression in CA1 pyramidal neurons had no effect on
glutamatergic synaptic transmission. Furthermore, we find that reducing bSAP97 expression
specifically within the dentate gyrus is sufficient to disrupt contextual episodic memory processing
in rats. Similar memory deficits are present in individuals with schizophrenia and have been
proposed to contribute to the development of delusions, disorganization, hallucinations, and the
loss of a sense of personal identity observed with this disorder (Servan-Schreiber et al., 1996,
Hemsley, 2005, Bazin et al., 2000, Rizzo et al., 1996a, Rizzo et al., 1996b, Waters et al., 2004,
24
Doughty et al., 2008, Murty et al., 2018, Aleman et al., 1999, Greenland-White et al., 2017,
Lewandowski et al., 2011, Doughty and Done, 2009, Libby et al., 2013). Finally, we show that
schizophrenia-related missense mutations clustered in SAP97’s PDZ2 domain also produce large
increases in synaptic AMPAR function in DG granule neurons that can be attributed to the release
of perisynaptic GluA1-containing AMPARs into the PSDs of perforant pathway synapses.
Altogether, our study identifies a cell-type specific synaptic regulatory mechanism in the dentate
gyrus that, when disrupted, impairs contextual episodic memory in rats.

25
2.3 Results

bSAP97 knockdown augments synaptic AMPAR-mediated neurotransmission in DG granule
neurons

Both dentate gyral and SAP97 dysfunction have been implicated in the development of
schizophrenia. To determine where bSAP97 is endogenously expressed in the rat hippocampus,
we performed an immunohistochemical analysis of bSAP97 expression in hippocampal slices
from rats. Remarkably, this analysis revealed robust expression of bSAP97 in the molecular layer
of the dentate gyrus but not in the stratum radiatum or stratum oriens of CA3 and CA1 (Fig. 2.1a).
bSAP97 expression overlapped with the dendritic-marker MAP2 in the molecular layer of the
dentate gyrus, indicating that bSAP97 is expressed in the dendrites of DG granule neurons (Fig.
2.1a and Supplementary Fig. 2.1a). We validated this experiment using a second SAP97 antibody,
which again displayed dendritic labeling that was specific to DG granule neurons (Supplementary
Fig. 2.1b). An isotype control primary antibody, as well as the secondary antibody alone, produced
no visible signal in our slices (Supplementary Fig. 2.1c). An immunizing peptide blocking
experiment was also performed to further validate the specificity of our immunolabeling, and again
no visible signal was found with the peptide blocked sample, demonstrating that the
immunolabeling was specific (Supplementary Fig. 2.1c). Together, these results suggest that
glutamatergic perforant pathway synapses between presynaptic entorhinal cortical neurons and
postsynaptic DG granule neurons may be selectively regulated by bSAP97 in DG granule neurons.
To determine whether bSAP97 plays a role in DG granule neuron glutamatergic synapse regulation,
we generated an RNAi against bSAP97. AAV-mediated expression of our bSAP97-microRNA
(miR) construct in dissociated rat hippocampal neurons reduced endogenous bSAP97 protein
26
levels by ~75% with no effects observed on the expression of other MAGUK proteins (Fig. 2.1b).
Using our miR construct, we knocked down expression of bSAP97 in DG granule neurons in rat
organotypic entorhino-hippocampal slice cultures through biolistic transfection (Schnell et al.,
2002, Elias et al., 2008). Biolistic transfection of these slices allows for sparse transfection of
neurons maintained within their intact, endogenous circuitry (Sadybekov et al., 2017, Tian et al.,
2018, Paskus et al., 2019, Rao et al., 2019, Herring et al., 2013, Herring and Nicoll, 2016). 6 days
after transfection, recordings of AMPA receptor- and NMDA receptor-evoked excitatory
postsynaptic currents (AMPAR- and NMDAR-eEPSCs) were made from fluorescent bSAP97-
miR transfected DG granule neurons and neighboring control neurons simultaneously during
perforant pathway stimulation (Fig. 2.1c). This approach allows for a pair-wise, internally
controlled comparison of the consequences of acute genetic manipulations that are limited to the
postsynaptic neuron (Sadybekov et al., 2017, Tian et al., 2018, Paskus et al., 2019, Rao et al., 2019,
Herring et al., 2013, Herring and Nicoll, 2016). Knocking down bSAP97 in DG granule neurons
using this approach led to a striking 4-fold increase in AMPAR-eEPSC amplitudes compared to
paired control neurons (Fig. 2.1d), a surprising phenotype given that knocking down traditional
MAGUK proteins reduces AMPAR-eEPSC amplitudes in CA1 pyramidal neurons (Levy et al.,
2015). A significant change in NMDAR-eEPSC amplitude was not observed following bSAP97
knockdown in DG granule neurons (Fig. 2.1e). RT-PCR analysis confirmed that the increase in
AMPAR-eEPSC amplitude we observed was not due to a secondary upregulation of aSAP97
expression (Supplementary Fig. 2.2a). aSAP97 protein is not endogenously expressed in the
hippocampus (Schluter et al., 2006) but has been shown to augment synaptic function when
exogenously expressed in neurons (Waites et al., 2009). To further verify that the 4-fold increase
in AMPAR-eESPC amplitude was indeed due to knockdown of bSAP97 specifically, we generated
27
a recombinant, RNAi-resistant bSAP97 expression construct (Supplementary Fig. 2.2b). We first
verified that the expression of this RNAi-resistant bSAP97 was not inhibited by our bSAP97-miR
(Supplementary Fig. 2.2c). Next, we co-expressed our bSAP97-miR with an mCherry-tagged
RNAi-resistant bSAP97, and verified via imaging that this recombinant bSAP97 localized to
dendritic spines, consistent with previous work demonstrating perisynaptic localization of bSAP97
(Waites et al., 2009) (Supplementary Fig. 2.2d). Finally, we co-expressed our bSAP97-miR and
the RNAi-resistant bSAP97 construct in DG granule neurons and found that expression of the
RNAi-resistant bSAP97 returned AMPAR-eEPSC amplitude to wild-type levels (Fig. 2.1d and
Supplementary Fig. 2.2e), confirming the specificity of our genetic manipulation.
The increase in synaptic AMPAR-eEPSC amplitude we observe following knockdown of
bSAP97 may arise from increased exocytosis of intracellular AMPARs or rearrangements of
AMPARs already on surface of neurons that result in more AMPARs in the PSD of glutamatergic
synapses. To determine whether bSAP97 supports intracellular AMPAR stores or inhibits surface
AMPARs from entering the PSD, we puffed glutamate onto the dendrites of neighboring whole-
cell patch clamped control and bSAP97-miR-expressing DG granule neurons. Puffing glutamate
onto the dendrites of these neurons produced currents stemming from the activation of
extrasynaptic, perisynaptic, as well as synaptic AMPARs on the surface of dendrites. This
approach allowed us to compare the number of AMPARs on the dendritic surface of neurons in
the presence and absence of bSAP97. We found that knocking down bSAP97 produced no change
in AMPAR surface current amplitude in DG granule neurons (Fig. 2.1f). These data are consistent
with that of previous work demonstrating that recombinant bSAP97 restricts the ability of
perisynaptic AMPARs on the spine surface from accessing the PSD of glutamatergic synapses
(Waites et al., 2009).
28
Given that bSAP97 likely prevents a subpopulation of AMPARs from reaching synapses, we
were interested in whether bSAP97 associates with AMPARs that have a subunit composition
distinct from synaptic AMPARs. Previous studies have reported the presence of calcium
permeable GluA2 subunit-lacking AMPARs in neurons that are made available to synapses during
activity dependent synaptic potentiation (Park et al., 2018). One possibility is that bSAP97 binds
to and holds GluA2-lacking AMPARs away from glutamatergic synapses in DG granule neurons,
and that knocking down bSAP97 releases these receptors, allowing them to reach synapses. To
test this hypothesis, we compared synaptic AMPAR rectification in control neurons to neurons
expressing our bSAP97-miR. GluA2-lacking AMPARs exhibit inwardly rectifying currents
whereas GluA2-containing AMPARs have a linear current/voltage relationship. We found that
knocking down bSAP97 had no effect on +40/-70 AMPAR-eEPSC amplitude ratios, with both
control neurons and bSAP97 knockdown neurons exhibiting linear +40/-70mV AMPAR-eEPSC
amplitude ratios characteristic of GluA2-containing AMPARs (Fig. 2.1g). Thus, we conclude that
knocking down bSAP97 does not result in the insertion of GluA2-lacking AMPARs into
glutamatergic synapses.
Finally, we performed additional basic characterizations of the effects of bSAP97
knockdown on other neuronal and synaptic properties. We determined that knockdown of bSAP97
has no effect on the resting membrane potential or the excitability of DG granule neurons
(Supplementary Fig. 2.2f). We also found that bSAP97 knockdown in DG granule neurons has no
effect on GABAergic synapse function (Fig. 2.1h).

29
bSAP97 knockdown has no effect on glutamatergic neurotransmission in CA1 pyramidal neurons

Our immunohistochemical analysis revealed no expression of bSAP97 in the dendrites of
CA1 pyramidal neurons (Fig. 2.2a and Supplementary Fig. 2.1b). However, bSAP97 expression
was observed in the cell bodies of these neurons. To determine whether inhibiting bSAP97
expression affects glutamatergic synaptic transmission in CA1 pyramidal neurons, we transfected
these neurons with our bSAP97-miR. We then examined AMPAR- and NMDAR-eEPSC
amplitudes in pairs of bSAP97-miR transfected and control CA1 pyramidal neurons following
Schaffer collateral stimulation (Fig. 2.2b). As suspected by the lack of bSAP97 expression in the
dendrites of these neurons, knocking down bSAP97 led to no significant effects on either AMPAR-
or NMDAR-eEPSC amplitudes (Fig. 2.2c, d). We also assessed the effects of bSAP97 knockdown
on dendritic AMPAR surface current in CA1 pyramidal neurons by puffing glutamate onto the
apical dendrites of neighboring whole-cell patch clamped control and bSAP97-miR-expressing
CA1 pyramidal neurons. We found that bSAP97 knockdown does not affect dendritic AMPAR
surface current in CA1 pyramidal neurons (Fig. 2e). In further characterizing the effects of bSAP97
knockdown in CA1 pyramidal neurons, we stimulated axons within the stratum oriens to evaluate
the function of glutamatergic synapses on basal dendrites (Supplementary Fig. 2.3a) and again
found no effect on either AMPAR- or NMDAR-eEPSC amplitudes (Supplementary Fig. 2.3b, c).
Taken together with our results in DG neurons, we have now identified a specific set of
glutamatergic synapses where bSAP97 plays an essential, cell type-specific role in regulating
glutamatergic neurotransmission.

30
PSD-95, PSD-93, SAP102 knockdown similarly affects CA1 pyramidal neurons and DG granule
neurons

In the hippocampus, our results demonstrate that bSAP97 inhibits synaptic AMPAR function
specifically in DG granule neurons. This is in contrast to PSD-95, PSD-93, and SAP102, which
are expressed in the dendrites of CA1 and CA3 pyramidal neurons and facilitate synaptic AMPAR
and NMDAR function in CA1 pyramidal neurons and many other neurons in the brain (Won et al.,
2017, Levy et al., 2015, Elias et al., 2006, El-Husseini et al., 2000, Beique and Andrade, 2003,
Ling et al., 2012, Cuthbert et al., 2007, Su et al., 2018). In addition to being expressed in the
dendrites of CA1 and CA3 pyramidal neurons, PSD-95, PSD-93, and SAP102 immunolabeling is
also present in the molecular layer of the dentate gyrus (Ling et al., 2012, Cuthbert et al., 2007, Su
et al., 2018). Therefore, we considered the possibility that all major MAGUK proteins play
fundamentally different roles in DG granule neurons compared to what has been conventionally
established in CA1 pyramidal neurons. If this was the case, it would suggest that the phenotype
we observed in DG granule neurons following bSAP97 knockdown is due to general MAGUK
function being different in DG granule neurons, rather than bSAP97 itself playing a unique
regulatory role at perforant path-DG granule neuron synapses. To test this, we compared the result
of knocking down PSD-95, PSD-93, and SAP102 in either CA1 or DG granule neurons by
expressing a previously-validated chained, triple MAGUK miR construct(Levy et al., 2015)
through biolistic transfection and stimulating Schaffer collaterals (Fig. 2.3a) or perforant pathways
(Fig. 2.3d), respectively. Similar to a previous report (Levy et al., 2015), we found that knocking
down PSD-95, PSD-93, and SAP102 in CA1 pyramidal neurons led to reductions in both AMPAR-
(Fig. 2.3b, g) and NMDAR-eEPSC amplitudes (Fig. 2.3c, h). When we repeated the experiment in
DG granule neurons, we found that the resulting reductions in AMPAR- and NMDAR-mediated
31
current amplitudes were nearly identical to the deficits in CA1 neurons following the same genetic
manipulation (Fig. 2.3e-h). Our observation of very similar synaptic phenotypes in CA1 and DG
granule neurons following knockdown of PSD-95, PSD-93, and SAP102 demonstrates that these
MAGUK proteins do not play a unique role in DG granule neurons in general. Rather, these data
demonstrate that bSAP97 plays a unique and vital synaptic regulatory role specifically in DG
granule neurons in a manner that is distinct from other MAGUKs.

Inhibition of bSAP97 function in the dentate gyrus disrupts contextual episodic memory

Microdeletion mutations resulting in reduced SAP97 expression have been linked to a 40-
fold increase in the risk of developing schizophrenia (Mulle et al., 2010, Mulle, 2015). Given that
reduced bSAP97 expression results in dramatic augmentation of glutamatergic synapse strength
in the DG granule neurons, we were interested in whether reduced bSAP97 expression within the
dentate gyrus produces behavioral phenotypes associated with schizophrenia. A favored cognitive
model of the abnormal behaviors and experiences characteristic of schizophrenia suggests that
they may be linked to a disturbance in the effects of context (Hemsley, 2005, Murty et al., 2018,
Aleman et al., 1999, Greenland-White et al., 2017, Lewandowski et al., 2011, Doughty et al., 2008,
Doughty and Done, 2009, Libby et al., 2013). More specifically, it has been suggested that
individuals with schizophrenia exhibit a diminished ability to assess the relative importance of
contextual cues in their environment, and that such a deficit may ultimately lead to the
development of delusions, disorganization, hallucinations, and the loss of a sense of personal
identity (Servan-Schreiber et al., 1996, Hemsley, 2005, Bazin et al., 2000, Rizzo et al., 1996a,
Rizzo et al., 1996b, Waters et al., 2004). A prediction of this model of schizophrenia is that the
normal effects of context on memory should be strongly reduced in patients with schizophrenia,
32
as this type of information is believed to be poorly integrated into the episodic representation
(Talamini et al., 2010). Thus, we were interested in whether reduced bSAP97 expression
specifically in the dentate gyrus produces a deficit in contextual information processing in rodents.
To determine whether diminished bSAP97 expression specifically within the dentate gyrus
affects the ability of animals to process contextual information, we performed bilateral stereotaxic
injections of our AAV virus containing the bSAP97-miR expression construct into either the
dentate gyrus or the hippocampal CA1 region of Sprague Dawley rats (Fig. 2.4a and
Supplementary Fig. 2.4). Following viral transduction, we examined contextual episodic memory
in these animals by assessing performance on a Novel Object in Context task (Davis et al., 2020,
Suarez et al., 2018, Noble et al., 2017). In this task, rats were acclimated to a unique combination
of objects in two different contexts, Context 1 and Context 2 (Fig. 2.4b). The animals were then
placed in Context 2 containing the combination of objects from Context 1. One of these objects
was previously unique to Context 1 and was novel in Context 2. Rats are capable of recognizing
this novel, “out of context”, object as evidenced by increased exploration time (Fig. 2.4b). Our
results revealed that compared to control animals, bSAP97-miR expression in the dentate gyrus
led to a significantly reduced time spent exploring the out of context object in Context 2, expressed
as a shift from baseline preference for the same object when presented in Context 1 (Fig. 2.4b; see
methods). In other words, animals with compromised bSAP97 expression in the dentate gyrus did
not appear to recognize this object as out of context. In marked contrast, expression of our
bSAP97-miR in the CA1 region of the hippocampus produced no effect on rat performance on the
Novel Object in Context task (Fig. 2.4b). To ensure that the decreased exploration times of the out
of context object we observed with bSAP97-miR expression in the dentate gyrus were not
secondary to a general avoidance of novel objects due to altered anxiety, we tested anxiety-like
33
behavior using the Zero Maze test. Results showed no differences between groups in time spent in
open zones, indicating no effect of dentate gyral bSAP97-miR expression on anxiety-related
behavior (Fig. 2.4c). We also observed no effect of dentate gyral bSAP97-miR expression on
locomotion as indicated by distance traveled in an open field (Fig. 2.4d). Furthermore, the inability
of animals to identify the out of context object following bSAP97-miR expression in the dentate
gyrus was not due to the inability of these animals to identify novel objects as evidenced by similar
performance to controls on a standard perirhinal cortex-dependent Novel Object Recognition task
(Fig. 2.4e). bSAP97-miR expression in the CA1 region of the hippocampus also did not affect
performance on the Zero Maze, Open Field or Novel Object Recognition tasks (Fig. 2.4c-e). Thus,
we conclude that compromised bSAP97 expression specifically within the dentate gyrus is
sufficient to produce a substantial deficit in contextual information processing in rats. These data
also demonstrate that disruption of bSAP97 function specifically within the dentate gyrus results
in behavioral consequences that are related to cognitive impairments observed in individuals with
schizophrenia.

Schizophrenia-related mutations in bSAP97 release GluA1-containing AMPARs into perforant
pathway synapses

Missense mutations in SAP97 have been identified in individuals with schizophrenia and are
clustered in SAP97’s PDZ2 domain (Fromer et al., 2014, Xing et al., 2016) (Fig. 2.5a). Two
unrelated individuals with schizophrenia have the same missense mutation in SAP97, SAP97-
G344R (Xing et al., 2016) (Fig. 2.5a). An additional individual with schizophrenia was also
identified harboring the de novo mutation SAP97-G357S (Fromer et al., 2014) (Fig. 2.5a).
34
SAP97 binds directly to AMPARs through an interaction between GluA1’s seven amino acid
C-terminal PDZ-binding domain and SAP97’s PDZ2 domain (Leonard et al., 1998, Sans et al.,
2001, Kim et al., 2005, Zhou et al., 2008, Cai et al., 2002) (Fig. 2.5a). This is in contrast to PSD-
95, PSD-93, and SAP102 which associate with AMPAR subunits though an intermediate
interaction with transmembrane AMPAR regulatory proteins (TARPs) (Jackson and Nicoll, 2011).
The structure of SAP97’s PDZ2 domain bound to GluA1’s C-terminal PDZ binding domain has
been solved(von Ossowski et al., 2006). Using this structure, our protein structural modeling
predicted that the schizophrenia-related missense mutations identified in SAP97’s PDZ2 domain
will inhibit SAP97’s ability to associate with GluA1’s PDZ-binding domain (Fig. 2.5b and
Supplementary Fig. 2.5). Our modeling was then validated using co-immunoprecipitation of
GluA1 with SAP97’s PDZ2 domain in HEK293T cells. As predicted, we found that schizophrenia-
related missense mutations in SAP97’s PDZ2 domain significantly inhibit binding to GluA1 (Fig.
2.5c and Supplementary Fig. 2.6a).
Based on our results demonstrating increased AMPAR-eEPSC amplitude following
knockdown of bSAP97 in DG granule neurons, we postulated that this increase in synaptic
AMPAR expression was a result of reduced interaction between bSAP97’s PDZ2 domain and
GluA1’s PDZ-binding domain. However, previous studies in CA1 pyramidal neurons have led
groups to conclude that GluA1’s PDZ-binding domain is dispensable for glutamatergic
neurotransmission (Granger et al., 2013, Kim et al., 2005). If disruption of a direct interaction
between GluA1 and bSAP97 is responsible for the increase in AMPAR-eEPSC amplitude that we
observe following knockdown of bSAP97 expression in DG granule neurons, then removing
GluA1’s PDZ-binding domain and preventing its ability to bind to bSAP97 should also increase
AMPAR-eEPSC amplitude. To test this idea, we employed a molecular replacement approach by
35
biolistically co-transfecting a previously-validated GluA1-shRNA construct (Lussier et al., 2014)
and a form of shRNA-resistant GluA1 lacking the C-terminal PDZ-binding domain (GluA1-D7)
(Fig. 2.5d) in both CA1 and DG granule neurons. Consistent with previous reports (Granger et al.,
2013, Kim et al., 2005), we found that molecular replacement of GluA1 with GluA1-D7 in CA1
pyramidal neurons has no effect on AMPAR-eEPSC amplitude (Fig. 2.5e, g). In marked contrast,
we found that molecular replacement of GluA1 with GluA1-D7 in DG granule neurons
phenocopied knockdown of bSAP97, producing a dramatic increase in AMPAR-eEPSC amplitude
(Fig. 2.5f, g). We also assessed whether replacement of GluA1 with GluA1-D7 occludes a further
increase in AMPAR-eEPSC amplitude produced by bSAP97 knockdown in DG granule neurons.
We found that further augmentation was indeed occluded (Supplementary Fig. 2.6b), suggesting
that the augmented synaptic strength following GluA1 replacement with GluA1-D7 and following
bSAP97 knockdown in DG granule neurons results from disruption of the same molecular
mechanism. Taken together, these data demonstrate that a direct interaction between GluA1’s
PDZ-binding domain and bSAP97 mediates bSAP97’s ability to inhibit synaptic AMPAR
expression in DG granule neurons. We hypothesized from these data that reducing bSAP97
expression releases AMPARs from perisynaptic regions which are in turn captured by traditional
MAGUKs at the synapse. If such was the case, knocking down these traditional MAGUK proteins
at the synapse would prevent the bSAP97 knockdown-mediated augmentation of DG granule
neuron synapse function. To test this, we co-expressed the triple MAGUK miR construct with our
bSAP97-miR in DG granule neurons, and, consistent with our hypothesis, we found that bSAP97
knockdown does not augment AMPAR-eEPSC amplitude in DG granule neurons on the PSD-95,
PSD-93, and SAP102 knockdown background (Supplementary Fig. 2.6c).
Together, our data suggest that schizophrenia-related missense mutations that inhibit the
36
ability of bSAP97’s PDZ2 domain to interact with GluA1 (Fig. 2.5c) should produce a potentially
pathological release of GluA1-containing AMPARs into the glutamatergic synapses of DG granule
neurons. Before directly testing whether schizophrenia-related missense mutations in bSAP97’s
PDZ2 domain influence synaptic AMPAR function, we first examined the expression of wild-type
bSAP97, bSAP97-G344R, and bSAP97-G357S. We observed levels of bSAP97-G344R and
bSAP97-G357S expression that were comparable to wild-type bSAP97 (Fig. 2.5h). Additionally,
we observed targeting of bSAP97-G344R and bSAP97-G357S to dendritic spines in DG granule
neurons like that observed with wild-type bSAP97 (Supplementary Fig. 2.6d, 2.2d). We then
examined AMPAR-eEPSC amplitude in DG granule neurons where endogenous bSAP97 was
molecularly replaced with bSAP97-G344R or bSAP97-G357S (Fig. 2.5a, i, j). In contrast to
molecular replacement with wild-type bSAP97 (Fig. 2.1d, 2.5j), we found that both schizophrenia-
related mutant forms of bSAP97 produced dramatic increases in synaptic AMPAR-eEPSC
amplitude in DG granule neurons like that seen with knockdown of bSAP97 and molecular
replacement of GluA1 with GluA1-D7 (Fig. 2.5i, j). Molecularly replacing bSAP97 with bSAP97-
G344R, which produced the largest increase in AMPAR-eEPSC amplitude of the two missense
mutations, was found to have no effect on dendritic AMPAR surface current (Supplementary Fig.
2.6e). As with reduced bSAP97 expression, such data demonstrate that the AMPAR-eEPSC
augmentation produced by these schizophrenia-related missense mutations results from
rearrangement of AMPARs on the dendritic surface of DG granule neurons. Finally, we performed
quantal analysis on our bSAP97 knockdown AMPAR-eEPSC data and on our AMPAR-eEPSC
data from molecular replacements with the schizophrenia-related mutants to determine whether
the augmented AMPAR-eEPSC amplitude in these conditions is caused by an increase in quantal
size or quantal content (Gray et al., 2011, Levy et al., 2015, Sadybekov et al., 2017, Tian et al.,
37
2018, Rao et al., 2019, Tian et al., 2021). We found that the augmentations in all three conditions
were caused by an increase in quantal size rather than quantal content (Supplementary Fig. 2.6f).
Thus, the increases in AMPAR-eEPSC amplitude we observe are due to increased AMPAR
expression at existing functional glutamatergic synapses rather than an increase in glutamatergic
synapse number.
Altogether, our data demonstrate that both reduced bSAP97 expression and schizophrenia-
related missense mutations in bSAP97’s PDZ2 domain compromise bSAP97’s interaction with
GluA1-containing AMPARs and, as a result, cause rearrangements of AMPARs present on the
dendritic surface of DG granule neurons that lead to increased AMPAR expression in the PSDs of
existing functional glutamatergic synapses. We observe robust localization of bSAP97 in the
spines of DG granule neurons that is consistent with previous work demonstrating a perisynaptic
localization of bSAP97 (Waites et al., 2009). Therefore, we believe the most likely explanation
for our findings is that bSAP97 maintains perisynaptic pools of AMPARs in DG granule neurons
(Fig. 2.5k), and that inhibiting bSAP97’s interaction with GluA1 by way of reduced bSAP97
expression or missense mutations in bSAP97’s PDZ2 domain causes AMPARs to translocate from
these perisynaptic regions to the PSD resulting in aberrant increases in perforant pathway synapse
strength (Fig. 2.5k). Such elevations in the strength of perforant pathway synapses likely disrupt
dentate gyral information processing and may contribute to the development of behavioral
phenotypes associated with schizophrenia.

38
2.4 Discussion

While the synaptic roles of most MAGUK proteins are well understood (i.e. PSD-95, PSD-
93, and SAP102), the role of SAP97 in regulating glutamatergic neurotransmission has remained
unclear. In previous studies, overexpression of recombinant SAP97 in neurons has led to mixed
and inconclusive results (Nakagawa et al., 2004, Schnell et al., 2002, Schluter et al., 2006, Li et
al., 2011, Ehrlich et al., 2007, Rumbaugh et al., 2003). It is difficult to derive meaningful
conclusions from such experiments given inconsistencies in the SAP97 splice variants used across
studies. Furthermore, overexpressing recombinant proteins in neurons that do not utilize the
protein endogenously may lead to aberrant protein function. Inhibiting SAP97 expression has also
been shown to produce a variety of seemingly contradictory glutamatergic synapse phenotypes
(Nakagawa et al., 2004, Li et al., 2011, Howard et al., 2010). Such discrepancies have likely arisen
at least in part because of the different neuronal preparations used. In the case of dissociated neuron
cultures, neurons lose specificity in their synaptic connections and instead form synapses with
neighboring neurons regardless of the original circuitry. In such artificial contexts, neurons may
develop synapses utilizing proteins they otherwise would not. Critical controls for SAP97 RNAi
usage, such as rescue experiments, are also missing in previous studies making it difficult to rule
out off-target effects. Thus, previous literature has led to considerable confusion regarding
SAP97’s role in synaptic regulation and, based on the absence of a synaptic phenotype in CA1
pyramidal neurons following knock out of SAP97 (Howard et al., 2010), groups have generally
concluded that SAP97 has no role in the regulation of glutamatergic synapse function (Won et al.,
2017).
Driven by the implication of dentate gyral and SAP97 dysfunction in schizophrenia, the
present study visually identified the perforant pathway-DG granule neuron synapse as where
39
SAP97 might play an important synaptic role due to the robust endogenous bSAP97 expression in
the dendrites of DG granule neurons. We determined that inhibiting bSAP97 function specifically
in DG granule neurons leads to a dramatic increase in AMPAR-mediated currents, signaling an
increase in synaptic AMPAR expression. In contrast, we observed no effects on synaptic
transmission following knockdown of bSAP97 in CA1 pyramidal neurons, as predicted from the
lack of dendritic expression of bSAP97 in CA1 neurons. Taken together, our data demonstrate that
bSAP97 plays a critical cell-type specific regulatory role at perforant pathway-DG granule neuron
synapses. Thus, we have identified a specific set of synapses where bSAP97 is essential for
regulating glutamatergic neurotransmission. In contrast to a previous study performed in
dissociated hippocampal neurons (Li et al., 2011), we find that bSAP97 does not play a significant
role in governing synaptic NMDAR function. The lack of an effect we observe on NMDAR
function is consistent with previous biochemical evidence suggesting that SAP97 does not interact
with NMDARs (Wyszynski et al., 1997, Leonard et al., 1998). One possible explanation for this
discrepancy is that formation of glutamatergic synapses between hippocampal neurons outside of
their native circuitry confers unique properties to bSAP97. Intriguingly, we also observe a laminar
pattern of bSAP97 immunolabeling within the molecular layer of the dentate gyrus that is
consistent with higher levels of bSAP97 expression in the synapses of the lateral perforant pathway
relative to the medial perforant pathway. Going forward, it will be interesting to determine if
bSAP97 inhibition of AMPAR-mediated synaptic transmission is greater at lateral perforant
pathway synapses compared to those of the medial perforant pathway.
Having identified the synapses where SAP97 plays a critical role, we began characterizing
the mechanism by which bSAP97 regulates synaptic AMPAR expression. We found through IV
rectification analysis that bSAP97 appears not to govern synaptic AMPAR subunit composition.
40
GABAergic neurotransmission and intrinsic excitability were also unaffected by bSAP97
knockdown in DG granule neurons. Furthermore, we determined through exogenous glutamate
application that while the synaptic AMPAR function increased following bSAP97 knockdown,
AMPAR expression on the surface of DG granule neuron dendrites did not change. This
demonstrates that the increase in synaptic AMPAR function we observe is not due to the exocytosis
of intracellular AMPARs but rather a reorganization of AMPARs on the dendritic surface (Fig.
2.5k). Additionally, we found that knocking down MAGUKs PSD-95, PSD-93, and SAP102
decreased synaptic transmission similarly in CA1 pyramidal neurons and DG granule neurons.
This observation ruled out the possibility that the dramatic increase in synaptic AMPAR
expression following bSAP97 knockdown in DG granule neurons was due to all MAGUK proteins
playing a fundamentally different role in the dentate gyrus. Thus, our data demonstrate that
bSAP97 is distinct from the other MAGUKs in its function.
In humans, microdeletion mutations involving the DLG1 gene are believed to lower bSAP97
expression and contribute to a greater than 40-fold increase in the risk of developing schizophrenia
(Mulle et al., 2010, Mulle, 2015). The development of schizophrenia-related disorders has also
been linked to disruption of dentate gyral information processing (Yun et al., 2016, Das et al., 2014,
Tamminga et al., 2010, Kawano et al., 2015, Ota et al., 2017, Falkai et al., 2016, Kirov et al., 2013,
Jaffe et al., 2020, Nakahara et al., 2019, Tavitian et al., 2019). Given that reduction of bSAP97
expression resulted in dramatic augmentation of glutamatergic synapse strength in DG granule
neurons, we were interested in whether reducing bSAP97 expression specifically within the
dentate gyrus is sufficient to produce behavioral phenotypes associated with schizophrenia. A
well-established model of schizophrenia suggests that deficits in processing contextual
information during episodic memory formation may underlie core phenotypes associated with
41
schizophrenia including delusions, disorganization, hallucinations, and the loss of a sense of
personal identity (Servan-Schreiber et al., 1996, Hemsley, 2005, Bazin et al., 2000, Rizzo et al.,
1996a, Rizzo et al., 1996b, Waters et al., 2004). Here, we investigated contextual episodic memory
formation in rats following the expression of our bSAP97-miR in in the dentate gyrus or the CA1
region of the hippocampus. We found that animals with bSAP97-miR expression in the dentate
gyrus exhibited substantial impairment in contextual information processing during episodic
memory formation. In marked contrast, bSAP97-miR expression in the CA1 region of the
hippocampus produced no effect on contextual information processing. Thus, contextual
information processing is only compromised by our bSAP97-miR in the brain region where we
observe dendritic expression of bSAP97 and where glutamatergic synapse function is augmented
when bSAP97 expression is knocked down. Together, our results establish that disruption of
bSAP97 function specifically within the dentate gyrus is sufficient to produce behavioral deficits
consistent with those observed in individuals with schizophrenia. Given that we see no change in
GABAergic neurotransmission or intrinsic excitability in DG granule neurons following
knockdown of bSAP97 expression, we believe that the augmented glutamatergic synapse strength
we observe in these neurons is likely responsible for the deficit in contextual information
processing in these animals. However, we do acknowledge that we cannot definitively exclude the
relevance of some yet-to-be-discovered role of bSAP97 in regulating DG neuron function.
Traditional MAGUK proteins (PSD-95, PSD-93, and SAP102) interact with AMPARs
through TARP proteins, which contain PDZ-binding motifs (Jackson and Nicoll, 2011). bSAP97
differs from these MAGUKs in that its PDZ2 domain can directly interact with the C-terminal
PDZ-binding domain of the AMPAR subunit GluA1 (Leonard et al., 1998, Sans et al., 2001, Kim
et al., 2005, Zhou et al., 2008, Cai et al., 2002). In humans, schizophrenia-related missense
42
mutations in SAP97 are clustered in SAP97’s PDZ2 domain and here we show that these mutations
inhibit GluA1 binding to SAP97’s PDZ2 domain. These data suggested that inhibition of SAP97’s
interaction with GluA1 may be responsible for the increased synaptic AMPAR function we
observe in DG granule neurons, which may, in turn, contribute to the development of
schizophrenia in humans. In contrast to traditional MAGUK proteins, bSAP97 possesses an N-
terminal L27 domain that causes bSAP97 to localize to perisynaptic regions in spines outside of
the PSD (Waites et al., 2009, Funke et al., 2005). Based on this previous work and our own
bSAP97 localization data, we hypothesized that endogenous bSAP97 may sequester GluA1-
containing AMPARs perisynaptically in DG granule neurons through a direct interaction between
bSAP97 and GluA1 subunits. Reduced bSAP97 expression or schizophrenia-related missense
mutations in bSAP97’s PDZ2 domain may disrupt the direct interaction between bSAP97 and
GluA1’s C-terminal PDZ-binding domain causing AMPARs to be released into the PSD. If this is
the case, removing GluA1’s PDZ-binding domain in DG granule neurons should result in elevated
synaptic AMPAR function like that produced by bSAP97 knockdown. Removal of GluA1’s PDZ-
binding domain (GluA1-D7) was previously examined in CA1 pyramidal neurons and produced
no effect on glutamatergic neurotransmission, suggesting that in these neurons a GluA1-bSAP97
interaction likely does not play a regulatory role (Kim et al., 2005). In fact, the relevance of
GluA1’s entire C-tail in the regulation of glutamatergic neurotransmission was recently called into
question (Granger et al., 2013). In agreement with these studies, we found that molecularly
replacing endogenous GluA1 with GluA1-D7 in CA1 pyramidal neurons produced no effect on
baseline glutamatergic neurotransmission. However, when repeating the experiment in DG granule
neurons, we found a dramatic increase in AMPAR-eEPSC amplitude that phenocopies what is
observed with knocking down bSAP97. We also found that GluA1-D7 molecular replacement in
43
DG granule neurons prevents further augmentation of synaptic strength by our bSAP97-miR.
Together, such data demonstrate that disruption of bSAP97’s interaction with GluA1’s C-terminal
PDZ binding domain produces the dramatic increases in AMPAR-eEPSC amplitude that we
observe in DG granule neurons (Fig. 2.5k).
Our protein structural modeling and co-immunoprecipitation data demonstrate that
schizophrenia-related mutations clustered in bSAP97’s PDZ2 domain inhibit GluA1’s ability to
bind to this domain. Such data led us to predict that these mutant forms of bSAP97 would produce
pathological augmentation of synaptic AMPAR function in DG granule neurons. Indeed, we find
that molecular replacement of bSAP97 with these schizophrenia-related mutant forms of bSAP97
produce dramatic augmentation of AMPAR-eEPSC amplitude in DG granule neurons like that
seen with bSAP97 knockdown and molecular replacement of GluA1 with GluA1-D7. Quantal
analysis performed on these data demonstrate that the increase in AMPAR-eEPSC amplitude that
we observe with these mutations is due to increased AMPAR expression at existing functional
glutamatergic synapses. This finding is in line with previous work showing that recombinant
bSAP97 accumulates in dendritic spines as they structurally mature (Lambert et al., 2017). We
also show that these mutations do not impact bSAP97 expression, targeting of bSAP97 to spines,
or dendritic AMPAR surface current. Altogether, our data suggest that bSAP97 binds to the C-
terminal PDZ domain of GluA1-containing AMPARs and, through this interaction, maintains a
perisynaptic surface pool AMPARs within the spines of DG granule neurons (Fig. 2.5k). This
mechanism is consistent with that proposed by a previous study performed utilizing recombinant
bSAP97 in neurons (Waites et al., 2009). Furthermore, our data suggest that when the interaction
between bSAP97 and GluA1 is disrupted, either by reducing bSAP97 expression or schizophrenia-
related missense mutations in bSAP97’s PDZ2 domain, AMPARs are released from perisynaptic
44
sites into the PSD causing a pathological increase in synaptic strength in DG granule neurons (Fig.
2.5k) that likely disrupts information processing in the dentate gyrus. Going forward, it will be
important to determine why DG granule neurons uniquely employ bSAP97 to sequester AMPARs
outside of the PSD.
In conclusion, our study identifies a cell-type specific synaptic regulatory mechanism in the
dentate gyrus that, when disrupted, impairs contextual information processing in rats. As a result,
restoring proper bSAP97 function and/or reducing AMPAR-mediated neurotransmission within
the dentate gyrus should be considered when designing potential therapeutic strategies for
individuals harboring pathological mutations in the DLG1 gene. In future studies, it will also be
important to determine whether similar synaptic pathology in the dentate gyrus is produced by
schizophrenia-related mutations in other genes. Information derived from such studies will be
instrumental in establishing how broadly applicable these therapeutic strategies might be in the
treatment of this psychiatric disorder.

45
2.5 Methods

Experimental Constructs

bSAP97-miR target sequence 5’ – TCTACTGGAGGGCTAAGGCCG – 3’ was embedded into an
emerald GFP (emGFP) sequence in a pFUGW expression vector to make a pFUGW-bSAP97-miR
construct. For the SAP97 rescue experiment, cDNA for RNAi-resistant human bSAP97 with
inserts I1B, I3, and I5 was obtained from GE Dharmacon (CloneId: 9053182) and cloned into NheI
and XmaI sites of a pCAGGS-IRES-mCherry expression vector. Schizophrenia-related missense
mutants bSAP97-G344R and bSAP97-G357S were made from this pCAGGS-bSAP97-IRES-
mCherry construct using overlap-extension PCR followed by In-Fusion cloning (Clontech).
mCherry-tagged bSAP97 constructs (wild-type, G344R and G357S) were made by deleting the
IRES element and fusing the mCherry onto the C-terminus of bSAP97 in the pCAGGS-bSAP97-
IRES-mCherry and mutant constructs. The chained, triple MAGUK miR construct was previously
validated (Levy et al., 2015) and was generously provided by Dr. Roger Nicoll. Rat GluA1 (flip-
type) construct pCAGGS-GluA1-IRES-GFP, pCAGGS-FLAG-GluA1, and the FHUGW-
GluA1shRNA construct (target sequence 5’ - GGAATCCGAAAGATTGGTT – 3’) were
generously provided by Dr. Katherine Roche (Lussier et al., 2014). An RNAi-resistant form of
GluA1 was generated by introducing five silent mutations within the shRNA target sequence via
overlap-extension PCR followed by In-Fusion cloning (Clontech). shRNA-resistant GluA1
missing the C-terminal PDZ-binding domain (GluA1-D7), specifically missing the last seven
amino acid residues PLGATGL, was generated also using the overlap-extension PCR and In-
Fusion methods. All plasmids were confirmed by DNA sequencing. Oligonucleotide sequences
used to generate experimental constructs are provided in Supplementary Table 1.
46
Immunohistochemistry

Experiments were performed in accordance with NIH Guidelines for the Care and Use of
Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use
Committee of the University of Southern California. Postnatal day 15 (P15) Sprague Dawley rats
of both sexes were transcardially perfused with 11ml of cold PBS and 25ml of cold 4% PFA in
PBS at a flow rate of 3ml/min. The hippocampi were immediately dissected and were post-fixed
overnight at 4°C in 4% PFA. After 3 brief washes in PBS, the hippocampi were sliced using a
vibratome at 100µm thickness. Slices were placed into 24-well culture plates containing PBS and
stained within the wells. Slices were blocked in PBST (PBS + 0.25% TritonX-100) with 10% Goat
Serum for 1 hour at room temperature, rinsed in PBST, and incubated with primary antibody
diluted in PBST overnight at 4°C. Then the slices were thoroughly washed in PBST and stained
with secondary antibody diluted in PBST for 2 hours at room temperature. Slices were then
mounted onto slides, dried for 15 minutes, and mounted with either Fluoromount-G
(SouthernBiotech, Cat#0100-01) or Fluoroshield with DAPI (Sigma Aldrich, Cat#F6057). For the
immunizing peptide block experiment, a blocking peptide matching the epitope sequence of the
rabbit anti-SAP97 antibody EEYRSKLSQTEDRQLRSS was synthesized (>98% purity). Diluted
SAP97 primary antibody was prepared as normal, the blocking peptide was added to the antibody
at a 10:1 ratio, and the mixture was incubated overnight at 4°C. This peptide blocked antibody was
used following the same staining protocol as detailed above. Antibodies used are as follows: rabbit
anti-SAP97 (1:1000, Invitrogen Cat#PA1-044, RRID: AB_2092021), mouse anti-MAP2 (1:1000,
Sigma Aldrich Cat#4403), rabbit IgG polyclonal isotype control antibody (1:1000, Abcam
Cat#171870), rabbit anti-SAP97 (100ug, custom made, YenZym Antibodies, epitope sequence:
DQSEQETSDADQ), goat anti-rabbit Alexa Fluor 555 (1:1000, Invitrogen Cat#A32732, RRID:
47
AB_2633281), goat anti-mouse Alexa Fluor 488 (1:1000, Invitrogen Cat#A-11001, RRID:
AB_2534069). Slides were imaged with a Keyence All-in-One Fluorescence Microscope BZ-
X800 with a 4x objective for whole hippocampal slice imaging and with Zeiss 880 Confocal
Microscope with 10x and 40x water-immersion objectives.

AAV Production

Our emGFP-bSAP97-miR expression construct was subcloned into AAV-bGH(+) and packaged
into an adeno-associated virus (AAV2; Vector Biolabs) under the control of a UbC promoter to
create the bSAP97 miR AAV (titer = 1.0e13 GC/ml). A scrambled miR, emGFP-expressing AAV2
downstream of a UbC promoter (titer = 2.1e13 GC/ml) was used as a control (Vector Biolabs).

Immunoblotting and Co-Immunoprecipitation

For knockdown experiments in primary rat hippocampal dissociated neurons, neurons were
prepared from E18.5 Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA)
of both sexes and transduced with either bSAP97-miR AAV or scrambled-miR AAV at DIV1.
Lysates were prepared at DIV14 in RIPA buffer containing protease inhibitor mix (ThermoFisher,
Halt Protease Inhibitor Cocktail). Proteins were resolved by SDS-PAGE. Following transfer,
membranes were cut and analyzed by western blot with antibodies against SAP97 (1:1000,
Invitrogen, Cat#PA1-741, RRID: AB_2092020), PSD-95 (1:1000, Millipore, Cat#637258), PSD-
93 (1:1000, Millipore, Cat#618436), SAP102 (1:1000, Biolegend, Cat#832004), and b-actin
(1:1000, Cell Signaling Technology, Cat#4970S). Goat anti-rabbit, HRP-linked secondary
antibody (1:10000, Cell Signaling Technology, Cat#7074S) or horse anti-mouse, HRP-linked
48
secondary antibody (1:10000, Cell Signaling Technology, Cat#7076S) were used for all
immunoblotting experiments described. For validating bSAP97 rescue by the miR-resistant
bSAP97 construct, HEK293 cells (ATCC, Cat#CRL-1573) cultured in DMEM supplemented with
10% FBS and 1% penicillin-streptomycin and maintained at 37°C and 5% CO2 were transfected
using Lipofectamine 2000 (Invitrogen, Cat#11668027) with the following constructs: GFP &
bSAP97, bSAP97-miR & bSAP97, or bSAP97-miR & bSAP97 miR-resistant. Cells were
harvested 72 hours post transfection. Lysates were prepared in RIPA buffer containing protease
inhibitor mix, and proteins were resolved by SDS-PAGE. Following transfer, membranes were cut
and analyzed by western blot with antibodies against SAP97 (1:500, Neuromab, Cat#75-030) and
b-actin (1:1000, Cell Signaling Technology, Cat#4970S). For schizophrenia mutant expression
experiments, HEK293 cells were transfected with cDNAs of bSAP97, bSAP97-G344R, or
bSAP97-G357S using Lipofectamine 2000 and harvested 72 hours post transfection. Lysates were
prepared in RIPA buffer containing protease inhibitor mix, and proteins were resolved by SDS-
PAGE. Following transfer, membranes were cut and analyzed by western blot with the same
SAP97 and b-actin antibodies and concentrations as the experiments described above.

For co-immunoprecipitation experiments, HEK293T cells (ATCC, Cat#CRL-3216) were co-
transfected with FLAG-GluA1 and wild-type GFP-SAP97-PDZ2, GFP-SAP97-PDZ2(G344R), or
GFP-SAP97-PDZ2(G357S) using Lipofectamine 2000. 24 hours following transfection, cells were
washed and lysed (lysis buffer: 25mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40,
5% glycerol). Lysates were rocked for 30 minutes at 4°C and centrifuged. Supernatants were
collected and incubated with anti-GFP antibody (mouse, Neuromab, Cat#75-131) overnight at 4°C.
Protein G Dynabeads (ThermoFisher, Cat#10007D) were added to the lysate/antibody mixture and
49
incubated at 4°C for 4 hours. Beads were washed and eluted in the Dynabead elution buffer at
room temperature to ensure that FLAG-GluA1 was detectable at the expected monomeric
molecular weight. Whole cell lysates used as inputs were boiled to improve resolution and band
signal for both FLAG-GluA1 and GFP-SAP97-PDZ2. Proteins were resolved by SDS-PAGE.
Following transfer, membranes were cut and analyzed by western blot with antibodies against
FLAG (1:2000, Sigma Aldrich, Cat#8592) and GFP (rabbit, 1:1000, Invitrogen, Cat#A-11122).
Blots were quantified using Fiji. To compare the interactions between FLAG-GluA1 and WT,
G344R, or G357S GFP-SAP97-PDZ2, bands representing immunoprecipitated GFP-SAP97-
PDZ2, co-immunoprecipitated FLAG-GluA1, and input FLAG-GluA1 were measured. The
fraction of input FLAG-GluA1 that was co-immunoprecipitated was calculated by dividing the co-
immunoprecipitated FLAG-GluA1 by input FLAG-GluA1. This number was normalized to
immunoprecipitated GFP-SAP97-PDZ WT, G344R, or G357S, resulting in the final numbers used
to compare WT, G344R, and G357S GFP-SAP97-PDZ2, respectively.

Real-Time Polymerase Chain Reaction

Rat hippocampal neurons were prepared from E18.5 Sprague Dawley rats (Charles River
Laboratories, Wilmington, MA, USA) of both sexes and transduced with either bSAP97-miR
AAV or scrambled miR AAV at DIV1. At DIV14 cells were suspended in a lysis buffer containing
1% beta-mercaptoethanol and disrupted using QIAshredder homogenizers (Qiagen, Cat#79654).
Total RNA was purified and isolated with the RNeasy Micro kit (Qiagen, Cat#74004) following
manufacturer’s instructions. Total RNA content was quantified using the Nanodrop One
spectrophotometer (ThermoFisher Scientific, Cat#ND-ONE-W) and the Quantitect Reverse
Transcription kit was employed to synthesize complimentary DNA from 500ng of total RNA
50
(Qiagen, Cat#205311). Real-time polymerase chain reaction (RT-PCR) was run on a QuantStudio
5 RT-PCR system (Applied Biosystems, Cat#A28140) using the Taqman Fast Advanced Master
Mix (Applied Biosystems, Cat#4444557) and Taqman Gene Expression Assay Mix for GAPDH
(Assay ID Rn01775763_g1), aSAP97 (Assay ID ART2CT4) and bSAP97 (Assay ID
Rn01439452_m1). CT values were obtained from the QuantStudio 5 Design & Analysis software
and converted to fold changes using the Delta-Delta CT method.

Electrophysiology

400 µm rat organotypic entorhino-hippocampal slice cultures were prepared from both male and
female P6 to P8 Sprague Dawley rats as previously described (Bonnici and Kapfhammer, 2009,
Prang et al., 2001, Stoppini et al., 1991). Hippocampi with accompanying entorhinal cortices were
removed from 6-10 rats at a time, and 400 μm transverse sections were made using a MX-TS tissue
slicer (Siskiyou). Slices from these 6-10 rats were mixed together and then mounted on individual
squares of Biopore Membrane filter roll (Millipore) and placed on Millicell Cell Culture inserts
(Millipore) in 35 mm dishes containing 1 ml of culture media (MEM + HEPES (Gibco Cat#12360-
038), horse serum 25%, HBSS (25%) and L-glutamine (1 mM). Media was exchanged every other
day. Slices with large portions of entorhinal cortex were visually identified subsequent to slicing.
These slices were selected and plated for use in our experiments, and the presence of entorhinal
cortex was again confirmed when selecting slices appropriate for data acquisition. Culture media
was exchanged every other day. Sparse biolistic transfections were performed on DIV1 as
described in detail previously(Schnell et al., 2002). 50ug total of mixed plasmid DNA was coated
on 1µm-diameter gold particles in 0.5mM spermidine, precipitated with 0.1mM CaCl2, and washed
four times in pure ethanol. These DNA-coated gold particles were then coated onto PVC tubing,
51
dried using ultra-pure N2 gas, and stored at 4°C in desiccant. Before use, the gold particles were
brought up to room temperature and then delivered to slice cultures via a Helios Gene Gun
(BioRad). For biolistically transfecting more than one construct, equal amounts of plasmid DNA
for each construct was used. Construct expression was confirmed by GFP or mCherry
epifluorescence. Electrophysiological recordings were performed on DIV7 slices. During
recordings, slices were maintained in room-temperature artificial cerebrospinal fluid (aCSF)
external solution containing (in mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose,
4 CaCl2, and 4 MgSO4. 5 µM 2-chloroadenosine and 0.1mM picrotoxin were also added to the
aCSF to dampen epileptiform activity and block GABAA receptor activity, respectively.
Osmolarity was adjusted to 310-315 mOsm. aCSF was saturated with 95% O2/5% CO2 throughout
recording. Borosilicate recording electrodes were filled with an internal, whole-cell recording
solution containing (in mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.3 EGTA, 5 QX-314, 4 Mg-
ATP, and 0.3 Na-GTP. The internal solution was adjusted to pH 7.3-7.4 and osmolarity of 290-
295 mOsm.

Transfected DG granule neurons and CA1 pyramidal neurons were identified using epifluoresence
microscopy. Dual whole-cell recordings of either neuronal subtype were made through
simultaneous recordings from one transfected neuron and a neighboring non-transfected control
neuron. Postsynaptic responses were evoked by stimulating with a monopolar glass electrode filled
with aCSF placed in the middle of the molecular layer to stimulate both medial and lateral perforant
pathway afferents for DG granule neuron recordings and at the stratum radiatum/stratum
lacunosum-moleculare border or in the stratum oriens for CA1 pyramidal neuron recordings. The
responses were acquired using a Multiclamp 700B amplifier (Molecular Devices), filtered at 2
52
kHz, and digitized at 10 kHz. AMPAR evoked EPSCS (eEPSCs) were measured at -70mV.
NMDAR-eEPSCs were measured at +40mV and were temporally isolated by measuring
amplitudes 150ms following the stimulus, at which point the AMPAR-eEPSC had completely
decayed. Data analysis was performed using Igor Pro (Wavemetrics). In the scatter plots for
simultaneous dual whole-cell recordings, each open circle represents one paired recording, and the
closed circle represents the average of all paired recordings. The diagonal line is shown on the
scatter plot to demonstrate that if the data point falls above the diagonal line, it indicates that the
eEPSC is lower in the control neuron, and vice versa. No more than one paired recording was
performed on any given entorhino-hippocampal slice. To elicit inhibitory synaptic responses, the
glass monopolar stimulating pipette was placed in the molecular layer of the dentate gyrus. 100
uM D-APV and 10 uM NBQX were added to a picrotoxin-free external solution to isolate
GABAR-eIPSCs.

For IV rectification analysis, 0.1 mM spermine was added to the internal solution described above
for measurement of AMPA receptor-mediated current rectification. Rectification indices were
calculated as the normalized glutamate-evoked current at +40 mV over −70 mV, respectively, in
presence of 100 μM APV to block NMDAR-mediated EPSCs.

For measuring surface AMPAR currents, a picospritzer II (General Valve Co.) was used to puff-
apply L-glutamate onto the dendrites of DG granule neurons in the molecular layer or onto the
apical dendrites of CA1 pyramidal neurons during recording. Glutamate pulses of 10 ms were
applied to patched neurons held at -70mV by a glass pipette. L-glutamate (25mM) was applied to
cells in a solution containing (in mM) NaCl 140, KCl 5, MgCl2 1.4, CaCl2 1, EGTA 5 and pH
53
adjusted to 7.4. 100 uM APV was added to isolate AMPAR currents.
For current-clamp recordings, the intracellular solution contained (in mM) 130 KMeSO4, 10 KCl,
10 HEPES, 4 NaCl, 1 EGTA, 4 Mg-ATP, and 0.3 Na-GTP. 500ms square current pulses were
delivered to neurons held in current clamp mode. For each recording, current pulse amplitude was
increased from 10 to 110 pA in 10 pA increments. Rheobase values were defined as the minimum
injected current required to elicit a single spike.

bSAP97 Localization Imaging

DG granule neurons in organotypic entorhino-hippocampal slice cultures made from P6-P8 rats
were biolistically co-transfected with mCherry-tagged bSAP97 constructs (wild-type, G344R, or
G357S) and the GFP expressing pFUGW-bSAP97-miR construct ~18-20 h after plating. Slices
were fixed in 4% PFA/4% Sucrose in PBS and washed 3x with PBS. Slices were further processed
with an abbreviated SeeDB-based protocol for imaging (Ke et al., 2013, Sadybekov et al., 2017,
Kay and Herring, 2021). Images were acquired at DIV7 via multiphoton confocal microscopy (SP8
LIGHTNING Confocal Microscope, Leica). Images were acquired using a 63x/1.4NA oil
immersion objective. Imaris image analysis software (Oxford Instruments) was used to identify
and visualize dendritic regions exhibiting the highest bSAP97 fluorescent intensity.

Protein Modeling

The high-resolution crystal structures of the SAP97 PDZ2 domain (PDB ID: 2AWX, 1.80Å
resolution; https://www.rcsb.org/structure/2AWX) and the SAP97 PDZ2 domain in complex with
a GluA1 C-terminal peptide (PDB ID: 2G2L, 2.35Å resolution) were used to predict the effect of
54
mutations on binding GluA1’s PDZ-binding domain. Calculations were performed using ICM
molecular modeling software (Molsoft LLC). Our modeling showed that mutation G357S results
in the formation of a hydrogen bond between hydroxyl group of S357 and carbonyl oxygen of
I354. Substitution of flexible Glycine to Serine and formation of an additional hydrogen bond
reduces the flexibility of the βC-αA loop. Moreover, this change may affect the conformational
changes of βA-βB observed upon peptide binding, as βA-βB and βC-αA loops are located in close
proximity to each other. The G344R mutation was predicted to impact the conformation of βB-
βC loop, as G344 has torsion angles that are not compatible with other amino acid residues.

Behavior – Animals and Surgery

For all behavior experiments, male Sprague Dawley rats (Envigo, Indianapolis, IN, USA)
weighing 300–400 g (~P90) were individually housed in wire-hanging cages in a climate
controlled (22–24 °C) environment with a 12:12 h light/dark cycle. Rats were given ad libitum
access to water and standard rodent chow (LabDiet 5001, LabDiet, St. Louis, MO). Experiments
were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals,
and all procedures were approved by the Institutional Animal Care and Use Committee of the
University of Southern California.

For stereotaxic injection of AAVs for in vivo knockdown of SAP97 expression in the dorsal
dentate gyrus (dDG) or in the dorsal CA1, rats were first anesthetized and sedated with a ketamine
(90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg) cocktail. Animals were then shaved
and the surgical site was prepped with iodine and ethanol swabs before being placed in a
stereotaxic apparatus for stereotaxic injections. bSAP97-miR AAV and scrambled-miR AAV were
55
prepared by Vector Biolabs, as described above. AAVs were delivered bilaterally to either the
dDG (AP: −3.12, ML: +/- 1.20, DV: −3.9) or the dorsal CA1 (AP: -3.12, ML: +/- 1.50, DV: -3.0)
at an injection volume of 200nl per hemisphere via pressure injections. Injections were
administered using a microinfusion pump (Harvard Apparatus, Holliston, MA) connected to a 33-
gauge microsyringe injector attached to a PE20 catheter and Hamilton syringe. Flow rate was
calibrated and set to 5μl/min. Injectors were left in place for 2 min to allow for complete infusion
of the drug. Behavioral experimental procedures began 21 days post virus injection to allow for
transduction and miR expression. Statistical analyses confirmed that the scrambled miR and
nonsurgical controls groups did not significantly differ for any behavioral measures, and thus these
groups were combined into a single control group for all subsequent analyses. Brains of all animals
that underwent behavioral testing were subjected to post hoc immunohistochemical analysis to
ensure that data was only included from animals where transduction was restricted to either the
dentate gyrus or CA1 region of the hippocampus (see Supplementary Fig. 2.4).

Novel Object in Context Task

Rats were tested on their episodic contextual memory abilities using the hippocampal-dependent
Novel Object in Context (NOIC) task, which was adapted from previous reports (Davis et al., 2020,
Suarez et al., 2018, Noble et al., 2017, Balderas et al., 2008, Martinez et al., 2014). Briefly, each
animal received a 5-min session per day in a behavioral box and the box and objects were cleaned
with 10% ETOH between each animal. Rats were habituated on consecutive days to Context 1, a
semi-transparent box (15in W × 24in L × 12in H) with yellow stripes, or Context 2, a grey opaque
box (17in W × 17in L × 16in H), counterbalanced by group and context order. Following
habituation, on day 1 of NOIC, each animal was placed in Context 1 containing a soap dispenser
56
(Object A) and an empty mason jar (Object B) placed on diagonal, equidistant markings with
ample space for the rat to circle the objects. Notably, the side the objects were located on (left or
right) was counterbalanced by group. The following day (day 2 of NOIC) rats were placed in
Context 2 placed in a different room with duplicates of either Object A or Object B. On the test
day, day 3 of NOIC, rats were placed again in Context 2, except this time with both Object A and
Object B. Depending on the duplicate objects seen on day 2 of NOIC, Object A or Object B on
day 3 of NOIC was not a novel object per se, but its placement in Context 2 was novel. Untreated
rats will preferentially explore the contextual novel object, an effect that would be disrupted with
hippocampal inactivation (Martinez et al., 2014). On day 1 and day 3, exploration was hand-scored
live by an experimenter blind to group assignments from video recordings using a ceiling Digital
USB 2.0 CMOS Camera (Stöelting Co., Wood Dale, IL) and was defined as sniffing or touching
the object with the nose or forepaws. Time spent exploring Novel Object in Context / (Time spent
exploring Object A + Object B)] x 100 was calculated on both days and the % shift from baseline
was determined by subtracting the value of day 1 from day 3.

Novel Object Recognition Task

NOR procedures were adapted from Beilharz et al (Beilharz et al., 2014). Briefly, a grey opaque
box (17in W x 17in L x 16in H) was used as an arena and placed in a dimly lit room, achieved by
pointing two desk lamps face down on opposite ends of the box. Rats were habituated to the empty
arena for 10 minutes on the day prior to testing. The novel object and the side on which the novel
object was placed was counterbalanced by group. The test began with a 5-minute familiarization
phase, where rats were placed in the center of the arena, facing away from the objects, with two
identical copies of the same object to explore. The objects were either two identical 12 oz. cans or
57
two identical stemless wine glasses, counterbalanced by treatment group. The objects were chosen
based on preliminary studies which determined that they are equally preferred by Sprague Dawley
rats. Animals were then removed from the arena and placed in the home cage for 5 minutes.
Meanwhile, the arena and objects were cleaned with 10% ethanol solution, and one of the objects
in the arena was replaced with a different one (either the 12 oz. can or stemless wine glass,
whichever the animal had not previously seen, i.e., the “novel object”). Then, the animals were
again placed in the center of the arena and allowed to explore for 3 minutes. Time spent exploring
the objects were hand-scored live from videos recorded from a ceiling Digital USB 2.0 CMOS
Camera (Stöelting Co., Wood Dale, IL). Then, the Recognition index [novel object exploration
(s)/[novel object exploration (s) + familiar object exploration (s)] was calculated for each animal,
with higher values on this index indicating greater exploration of the novel object.

Zero Maze Task

Rodents were tested for potential anxiety-like effects associated with injecting the bSAP97-miR
AAV into either the dorsal dentate gyrus or dorsal CA1 using a Zero Maze. The Zero Maze is an
elevated circular platform (63.5 cm height, 116.8cm external diameter) with two closed zones and
two open zones, all of which are equal in length. The closed zones are enclosed with 17.5 cm high
walls whereas the open zones have only 3 cm high curbs. Any-Maze software (Stöelting Co., Wood
Dale, IL) was used to video record the animals and analyze time spent in the open zones of the
maze. Animals were placed in the maze for a single 5 min session and after each session, the
apparatus was cleaned with 10% ethanol.

58
Open Field

Open field measures both general locomotor activity and anxiety-like behavior in the rat. Here, a
large grey bin, 60 cm (L) X 56 CM (W) was used as an arena and was placed under diffuse even
lighting (30 lux). A center zone (19 cm L X 17.5 cm W) was identified and marked using Any-
Maze software (Stoelting Co., Wood Dale, IL) and a USB camera directly overhead connected to
Any-Maze software tracked the movement of the animals. Animals were placed in the center of
the box and allowed to explore the arena for 10 min, with the dependent variable being the total
distance traveled (m). The apparatus was cleaned with 10% ethanol after each rat was tested.

Statistical Analysis

Paired electrophysiological recordings of eEPSC amplitudes were analyzed using paired two-
tailed T-tests. Two-sample two-tailed T-tests were used to compare electrophysiological data
across independent conditions. P-values of <0.05 were considered statistically significant
(KaleidaGraph). Coefficient of Variation (CV) analysis was performed on AMPAR-eEPSCs by
comparing the change in eEPSC variance with the change in mean amplitude as previously
described (Bekkers and Stevens, 1990, Malinow and Tsien, 1990, Gray et al., 2011, Levy et al.,
2015, Sadybekov et al., 2017, Tian et al., 2018, Rao et al., 2019, Tian et al., 2021). CV was
calculated as SD/M (SD = standard deviation; M = mean). The SD and M were measured,
normalized and plotted for a concurrent set of stimuli from a control and its neighboring transfected
cell. It has been shown both theoretically and experimentally that changes in CV
-2
(M
2
/SD
2
) are
independent of quantal size but vary in a predictable manner with quantal content: number of
release sites n x presynaptic release probability, Pr; CV
-2
= nPr/(1 – Pr) (Del Castillo and Katz,
59
1954, Bekkers and Stevens, 1990, Malinow and Tsien, 1990, Xiang et al., 1994). CV analysis is
presented as scatterplots with CV
-2
values calculated for transfected cell/control cell pairs on the
y-axis and mean eEPSC amplitude values of transfected/cell control cell pairs on the x-axis. Filled
circles represent the mean ± SEM of the entire dataset. Filled circles that fall on or near the 45° (y
= x) line reflect changes in quantal content while values approaching the horizontal (y = 1) reflect
a change in quantal size. Slice immunolabeling results were replicated using slices from at least
five different animals. Quantified biochemical results were replicated at least three times using
independent samples. For behavioral experiments, NOIC and NOR were analyzed using a multi-
factor ANOVA (Statistica Version 7; Statsoft) with surgical group, training squad, and novel
object assignment as between-subjects variables. All other behavioral tasks were analyzed using a
Student’s two-tailed two sample T-test performed using Statistica V7. The Grubbs test for outliers
was used as pre-established exclusion criteria (Prism 8). P-values of < 0.05 were considered
statistically significant. Error bars represent standard error of the mean measurement. Sample sizes
in the present study are similar to those reported in the literature (Incontro et al., 2018, Herring
and Nicoll, 2016) for electrophysiological recordings of eEPSC amplitudes and, for behavioral
tasks, sample size was chosen based on a priori power analyses (conducted in Statistica V7) to
ensure sufficient power to detect a pre-specified effect size. For biochemical data, two-sample T-
tests were used to compare experimental conditions.

60

Figure 2.1: bSAP97 knockdown augments AMPAR-mediated neurotransmission in DG
granule neurons
a (Left) Representative immunolabeling of bSAP97 in a rat entorhino-hippocampal slice. Red box
shows enlarged portion of the dentate gyrus. GL, granule layer; ML, molecular layer. (Right) Co-
61
immunolabeling of MAP2 and bSAP97 in dendrites of DG granule neurons. b Western blot
analysis showing specificity of bSAP97-miR in dissociated hippocampal neurons. bSAP97-miR
reduces bSAP97 protein expression in dissociated hippocampal neurons without altering PSD-95,
PSD-93, and SAP102 protein expression. (Right) Bar graph shows quantification of SAP97, PSD-
95, PSD-93, and SAP102 protein expression following bSAP97-miR-mediated knockdown. n
represents independent experiments. (bSAP97, n = 4, p = 0.00002; PSD-95, n = 3, p = 0.75; PSD-
93, n = 3, p = 0.62; SAP102, n = 3, p = 0.09; two sample T-tests). c Schematic representation of
electrophysiological recording setup for DG granule neurons. For panels (d) and (e), open circles
are single pairs of control and transfected neurons, filled circles represent the mean amplitudes
(±SEM), insets show representative current traces from control (black) and bSAP97-miR
transfected (green) neurons with stimulation artifacts removed. Scale bars: 20ms for AMPA, 50ms
for NMDA, 100pA. Bar graphs show the average AMPAR-eEPSC and NMDAR-eEPSC
amplitudes (±SEM) of DG granule neurons expressing the bSAP97-miR (green) and DG granule
neurons co-expressing the bSAP97-miR and miR-resistant wild-type (wt) bSAP97 cDNA (grey)
normalized to their respective control cell average eEPSC amplitudes (black). bSAP97-miR
expression increases AMPAR-eEPSC amplitude in DG granule neurons (n = 8 pairs, p = 0.019,
paired T-test) (d) but has no detectable effect on NMDAR-eEPSC amplitude (n = 7 pairs, p = 0.27,
paired T-test) (e). Co-expression of bSAP97-miR and miR-resistant bSAP97 cDNA has no
detectable effects on either AMPAR-eEPSC (n = 8 pairs, p = 0.227, paired T-test) (d) or NMDAR-
eEPSC amplitudes (n = 8 pairs, p = 0.99, paired T-test) (e). Additional details of the rescue
experiment are provided in Supplementary Fig. 2.2b, c, & e. f bSAP97-miR expression does not
change surface AMPAR current amplitude in DG granule neurons. (Left) Open circles in the
scatterplot represent single pairs of control and transfected neurons, filled circles represent the
62
mean amplitudes (±SEM), insets show representative surface AMPAR current traces from control
(black) and transfected (green) neurons. Scale bars: 5s, 25pA. (Right) Bar graph shows average
surface AMPAR current amplitudes (±SEM) of control (black) and bSAP97-miR expressing
(green) DG granule neurons (n = 5 pairs, p = 0.335, paired T-test). g bSAP97 knockdown does not
change AMPAR-eEPSC rectification. (Left) Representative current traces, scale bars: 20ms, 20pA.
(Right) Bar graph shows mean ± SEM of the AMPAR-eEPSC rectification index of control (black,
n = 5 neurons) and bSAP97-miR expressing (green, n = 5 neurons) DG granule neurons (p = 0.81,
two sample T-test). h bSAP97 knockdown does not affect GABAergic synapse function in DG
granule neurons. Open circles are single pairs of control and transfected neurons, filled circles
represent the mean amplitudes (±SEM), insets show representative current traces from control
(black) and bSAP97-miR transfected (green) neurons with stimulation artifacts removed. Scale
bars: 100pA, 100ms. Bar graph shows the average GABAR-eIPSC amplitudes (±SEM) of DG
granule neurons expressing the bSAP97-miR (green) and control DG neurons (black) (n = 5 pairs,
p = 0.81, paired T-test). *p < 0.05; n.s., not significant. All statistical tests performed were two-
tailed.
63

a (Left) Representative immunolabeling of bSAP97 in a rat entorhino-hippocampal slice. Blue
box shows enlarged CA1 region. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.
(Right) Co-immunolabeling of MAP2 and bSAP97 in dendrites of CA1 neurons. b Schematic
representation of electrophysiological recording setup for CA1 pyramidal neurons. For panels (c)
and (d), open circles are single pairs of control and transfected neurons, filled circles represent the
mean amplitudes (±SEM), insets show representative current traces from control (black) and
transfected (green) neurons with stimulation artifacts removed. Scale bars: 20ms for AMPA, 50ms
for NMDA, 20pA. Bar graphs show the average AMPAR-eEPSC and NMDAR-eEPSC
amplitudes (±SEM) of CA1 pyramidal neurons expressing the bSAP97-miR (green) and control
Figure 2.2: bSAP97 knockdown has no effect on glutamatergic neurotransmission in CA1
pyramidal neurons.
64
cell average eEPSC amplitudes (black). bSAP97-miR expression has no detectable effects on
either AMPAR-eEPSC (n = 7 pairs, p = 0.29, paired T-test, n.s., not significant) (c) or NMDAR-
eEPSC amplitudes (n = 6 pairs, p = 0.29, paired T-test, n.s., not significant) (d) in CA1 pyramidal
neurons. e bSAP97-miR does not change surface AMPAR current amplitude in CA1 pyramidal
neurons. (Left) Open circles in the scatterplot represent single pairs of control and transfected
neurons, filled circles represent the mean amplitudes (±SEM), insets show representative surface
AMPAR current traces from control (black) and transfected (green) neurons. Scale bars: 5s, 200pA.
(Right) Bar graph shows average surface AMPAR current amplitudes (±SEM) of control (black)
and bSAP97-miR expressing (green) CA1 pyramidal neurons (n = 5 pairs, p = 0.92, paired T-test).
n.s., not significant. All statistical tests performed were two-tailed.
65

a Schematic representation of electrophysiological recording setup for CA1 pyramidal neurons.
For panels b, c, e and f, open circles are single pairs of control and transfected neurons, filled
circles represent the mean amplitudes (±SEM), insets show representative current traces from
control (black) and transfected (green) neurons with stimulation artifacts removed. Scale bars: 20
ms for AMPA, 50ms for NMDA, 20 pA. b PSD-95, PSD-93, and SAP102 knockdown
Figure 2.3: PSD-95, PSD-93, SAP102 knockdown similarly affects CA1 pyramidal neurons
and DG granule neurons.
66
significantly decreases AMPAR-eEPSC amplitude in CA1 pyramidal neurons (n = 8 pairs, p =
0.00006, paired T-test). c PSD-95, PSD-93, and SAP102 knockdown significantly decreases
NMDAR-eEPSC amplitude in CA1 pyramidal neurons (n = 6 pairs, p = 0.03, paired T-test). d
Schematic representation of electrophysiological recording setup for DG granule neurons. e PSD-
95, PSD-93, and SAP102 knockdown significantly decreases AMPAR-eEPSC amplitude in DG
granule neurons (n = 7 pairs, p = 0.03, paired T-test). f PSD-95, PSD-93, and SAP102 knockdown
significantly decreases NMDAR-eEPSC amplitude in DG granule neurons (n = 6 pairs, p = 0.045,
paired T-test). g, h Summary bar graphs show the average AMPAR-eEPSC amplitudes (±SEM)
(g) and NMDAR-eEPSC amplitudes (±SEM) (h) of CA1 pyramidal neurons and DG granule
neurons expressing PSD-95, PSD-93, SAP102 miRs (green) normalized to their respective control
cell average eEPSC amplitudes (black). Two sample T-tests were used to compare across
independent conditions (CA1 vs. DG); (CA1 vs. DG (AMPA), n = 8 CA1 pairs, 7 DG pairs, p =
0.12; CA1 vs. DG (NMDA), n = 6 CA1 pairs, 6 DG pairs, p = 0.34). *p < 0.05; n.s., not significant.
All statistical tests performed were two-tailed.
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a Illustration of method used to inhibit bSAP97 function within the dentate gyrus or the CA1
region of the hippocampus. b (Left) Experimental setup of the Novel Object in Context behavioral
procedure. (Center) Compared to control animals, bSAP97-miR expression in the dentate gyrus
Figure 2.4: Knockdown of bSAP97 expression in the dentate gyrus disrupts contextual
episodic memory.
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led to a significantly reduced exploration of the object that was novel in Context 2, expressed as a
shift from baseline exploration for the same object in Context 1 (control n = 9, bSAP97 KD n =
12, p = 0.04, ANOVA). (Right) bSAP97-miR expression in the CA1 region led to no significant
changes in exploration of the object that was novel in Context 2 (control n = 8, bSAP97 KD n =
6, p = 0.61, ANOVA). c Control and bSAP97 knockdown animals spent similar amounts of time
in the open zones in a Zero Maze test for anxiety-like behavior following bSAP97-miR AAV
injections in either the dentate gyrus (Left, control n = 10, bSAP97 KD n = 12, p = 0.91, two
sample T-test) or the CA1 region (Right, control n = 8, bSAP97 KD n = 8, p = 0.3, two sample T-
test). d Control and bSAP97 knockdown animals traveled similar distances in an Open Field test
for exploratory behavior and general activity following bSAP97-miR AAV injections in either the
dentate gyrus (Left, control n = 10, bSAP97 KD n = 12, p = 0.2, two sample T-test) or the CA1
region (Right, control n = 9, bSAP97 KD n = 7, p = 0.76, two sample T-test). e Recognition
indices of control and bSAP97 knockdown animals were similar during a Novel Object
Recognition task following bSAP97-miR AAV injections in either the dentate gyrus (Left, control
n = 9, bSAP97 KD n = 12, p = 0.43, ANOVA) or the CA1 region (Right, control n = 9, bSAP97
KD n = 8, p = 0.54, ANOVA). *p < 0.05; n.s., not significant. All statistical tests performed were
two-tailed.

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a Illustration of bSAP97’s protein domain structure and location of schizophrenia-related missense
mutations identified in SAP97 (Top). Illustration of interaction between GluA1’s PDZ binding
domain and bSAP97’s PDZ2 domain (Bottom, dashed box). b Protein structural modeling of
schizophrenia-related mutations in bSAP97’s PDZ2 domain predict disruption of PDZ2’s
interaction with GluA1’s PDZ-binding domain (see Supplementary Fig. 2.5 for more details). c
Co-immunoprecipitation of FLAG-GluA1 with GFP-SAP97-PDZ2, GFP-SAP97-PDZ2(G344R),
Figure 2.5: Clustered schizophrenia-related missense mutations in bSAP97’s PDZ2 domain
release GluA1-containing AMPARs into perforant pathway synapses.
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or GFP-SAP97-PDZ2(G357S) in HEK293T cells. See methods for details. Quantification for n =
3 independent experiments provided in Supplementary Fig. 2.6a. d Topology of the AMPAR
subunit GluA1 and C-terminal amino acid sequences for GluA1 and GluA1-D7. GluA1’s PDZ
binding domain (PDZ BD) is highlighted in blue. e, f, and i Open circles in the scatterplots
represent single pairs of control and transfected neurons, filled circles represent the mean
amplitudes (±SEM), insets show representative current traces from control (black) and transfected
(in color) neurons with stimulation artifacts removed. Scale bars: 20ms, 20pA. e Molecular
replacement by co-expression of the GluA1-shRNA and GluA1-D7 has no effect on AMPAR-
eEPSCs in CA1 pyramidal neurons (n = 7 pairs, p = 0.16, paired T-test). f Molecular replacement
by co-expression of GluA1-shRNA and GluA1-D7 significantly increases AMPAR-eEPSC
amplitude in DG granule neurons (n = 7 pairs, p = 0.016, paired T-test). g Summary bar graph for
(e) and (f) shows the average AMPAR-eEPSC amplitudes (±SEM) of CA1 pyramidal neurons and
DG granule neurons with co-expression of GluA1-shRNA and GluA1-D7 normalized to their
respective control neurons. h Representative western blot analysis showing that bSAP97-G344R
and bSAP97-G357S exhibit levels of expression in HEK293 cells similar to that of wild-type
bSAP97 (n = 2 independent experiments). i Molecular replacement of bSAP97 with bSAP97-
G344R and bSAP97-G357S in DG granule neurons significantly increases AMPAR-eEPSC
amplitude (bSAP97-G344R, n = 7 pairs, p = 0.0004, paired T-test; bSAP97-G357S, n = 7 pairs, p
= 0.01, paired T-test). j Summary bar graph shows the average AMPAR-eEPSC amplitudes
(±SEM) of the conditions compared in panel i. *p < 0.05. k Model of bSAP97-mediated regulation
of glutamatergic synaptic function. GluA1-containing AMPARs on the surface of neurons are held
perisynaptically by an interaction between bSAP97 and GluA1’s PDZ-binding domain. Reducing
bSAP97 expression or inhibiting GluA1’s ability to bind to bSAP97’s PDZ2 domain results in
71
local rearrangement of AMPA receptor organization in spines whereby AMPA receptors normally
held by bSAP97 perisynaptically are released and then grabbed by PSD-95, PSD-93 and SAP102
in the PSD. All statistical tests performed were two-sided.

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a Immunolabeling of MAP2 and bSAP97 in a P15 rat hippocampal slice. Immunolabeling of
MAP2 and bSAP97 in dendrites of dentate gyrus and CA1 regions of the hippocampus shown in
Supplementary Figure 2.1: bSAP97 immunolabeling in the hippocampus is specific and
overlaps with MAP2 in DG granule neurons.

73
Fig. 2.1 were taken from regions shown as white squares in the merged image. b Immunolabeling
of bSAP97 in a P15 rat hippocampal slice using a second SAP97 antibody with an epitope distinct
from that used in a and Fig. 2.1a. Enlarged regions highlight SAP97 expression in DG granule
neuron dendrites but not in CA1 pyramidal neuron dendrites. ML, molecular layer; GL, granule
cell layer. c Immunolabeling with the bSAP97 antibody used in a and Fig. 2.1a, the rabbit
polyclonal primary antibody as isotype control, the secondary antibody only, and the bSAP97
antibody subsequent to peptide block with corresponding range indicator images for expression
levels.

74
a Average bSAP97 and aSAP97 mRNA expression (±SEM) in dissociated hippocampal neurons
transduced with AAVs expressing the bSAP97-miR or a scrambled miR. (Left: n = 3 scrambled
wells and 3 bSAP97-miR wells, p = 0.02; Right: n = 3 scrambled wells and 3 bSAP97-miR wells,
p = 0.11, two sample T-tests). b A cDNA expressing bSAP97 with splice inserts I1b, I3, I5 was
used in the bSAP97 molecular replacement experiments in the present study. c Immunoblot
Supplementary Figure 2.2: Supporting data for Figure 2.1
75
showing insensitivity of the miR-resistant bSAP97 rescue construct to the bSAP97-miR in
HEK293 cells (n = 1 experiment). Wild-type bSAP97 co-expressed with the bSAP97-miR leads
to reduced expression of bSAP97 (middle lane) compared to the bSAP97 + GFP control (left lane).
miR-resistant bSAP97 co-expressed with bSAP97-miR rescues this deficit (right lane) and leads
to similar levels of bSAP97 expression compared to the control. d Imaging experiment showing
synaptic localization of bSAP97-mCherry in a GFP-filled DG granule neuron in a cultured
entorhino-hippocampal slice. e Open circles represent single pairs of control and transfected
neurons, filled circles are the mean ±SEM, inset representative current traces are from control
(black) and transfected (green) neurons with stimulation artifacts removed. e Scatter plots for
bSAP97 wt rescue experiment (grey bar) in Fig. 2.1d, e. AMPAR-eEPSC: n = 8 pairs; NMDAR-
eEPSC: n = 8 pairs. Scale bars: 20ms for AMPA, 50ms for NMDA, 20pA. f bSAP97-miR
expression has no effect on the excitability or the resting membrane potential of DG granule
neurons. (Left) Representative traces of action potentials following current injection into bSAP97-
miR expressing (green) and control (black) DG granule neurons. (Center) Average number of
action potentials (±SEM) produced with injected current steps of increasing amplitude in bSAP97-
miR expressing and control DG granule neurons. (Right) Bar graphs showing that bSAP97-miR
expression has no effect on the rheobase (Rheobase; control n = 5 neurons, bSAP97-miR n = 6
neurons, p = 0.99, two sample T-test) nor on the resting membrane potential (Vrest; control n = 5
neurons, bSAP97-miR n = 6 neurons, p = 0.89, two sample T-test). *p < 0.05; n.s., not significant.
All statistical tests performed were two-tailed.

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a Schematic representation of electrophysiological recording setup for CA1 pyramidal neurons
following stimulation of axons within the stratum oriens. For b and c, open circles are single pairs
of control and transfected neurons, filled circles represent the mean amplitudes (±SEM), insets
show representative current traces from control (black) and bSAP97-miR transfected (green)
neurons with stimulation artifacts removed. Scale bars: 20ms for AMPA, 50ms for NMDA, 20pA.
Bar graphs show the average AMPAR-eEPSC and NMDAR-eEPSC amplitudes (±SEM) of CA1
pyramidal neurons expressing the bSAP97-miR (green) and control CA1 pyramidal neurons
(black). bSAP97-miR expression has no effect on neither AMPAR-eEPSC amplitude (n = 5 pairs,
p = 0.73, paired T-test) b nor NMDAR-eEPSC amplitude (n = 5 pairs, p = 0.94, paired T-test) c in
CA1 pyramidal neurons following stimulation of axons within the stratum oriens. n.s., not
significant. All statistical tests performed were two-tailed. Source data are provided in the Source
Data file.

Supplementary Figure 2.3: Supporting data for Figure 2.2
77

Representative coronal hippocampal sections are shown that were taken from rats with AAV-
bSAP97-miR injected into either the dentate gyrus a or CA1 region b subsequent to behavioral
testing. Fluorescence from the GFP expressed by the AAV-bSAP97-miR construct was enhanced
using immunohistochemistry. a Highly localized transduction of the dentate gyrus following
Supplementary Figure 2.4: Stereotaxic injection of the AAV-bSAP97-miR into the dentate
gyrus or CA1 of rats produced highly localized transduction within each region.
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stereotaxic injection of the AAV-bSAP97-miR. DGcr = dentate gyrus, crest; DGmb = dentate
gyrus, medial blade; DGlb = dentate gyrus, lateral blade; mo = dentate gyrus, molecular layer; sg
= dentate gyrus, granule cell layer; po = dentate gyrus, polymorph layer; hf = hippocampal fissure.
b Highly localized transduction of the CA1 region following stereotaxic injection of the AAV-
bSAP97-miR. CA1so = CA1, stratum oriens; CA1sp = CA1, stratum pyramidale; CA1sr = CA1,
stratum radiatum.

79

a Superimposition of the crystal structures of SAP97’s PDZ2 domain (grey) in complex with the
GluA1 C-terminal peptide (magenta) (PDBID: 2G2L) and structural models of wild-type SAP97’s
PDZ2 domain (blue) and G357S mutant (orange) with optimized βC-αA loop. Residue G357 is
shown as blue spheres and G357S is shown as orange spheres. b Magnified region of βC-αA loop
shows the mutated G357 residue in sticks (crystal structure in white, wild-type model in blue and
model of G344R mutant in orange). A hydrogen bond is shown as a blue dotted line. Our modeling
showed that the mutation G357S results in the formation of a hydrogen bond between the hydroxyl
group of S357 and the carbonyl oxygen of I354. Substitution of flexible Glycine to Serine and
formation of an additional hydrogen bond reduces the flexibility of the βC-αA loop. Moreover,
this change will likely affect the conformational changes of βA-βB observed upon peptide
binding
73
, as βA-βB and βC-αA loops are located in close proximity to each other. c
Supplementary Figure 2.5: Schizophrenia-related mutations in SAP97’s PDZ2 domain are
predicted to impact binding to GluA1’s PDZ-binding domain.
80
Superimposition of the crystal structures of SAP97’s PDZ2 domain (grey) in complex with the
GluA1 C-terminal peptide (magenta) and structural models of wild-type SAP97’s PDZ2 domain
(blue) and G344R mutant (orange) with optimized βB-βC loop. Residue G344 is shown as blue
spheres and G344R is shown as orange spheres. d Magnified region of βB-βC loop shows the
mutated G344 residue in sticks (crystal structure in white, wild-type model in blue and model of
G344R mutant in orange). Hydrogen bonds are shown as blue dotted lines. The G344R mutation
was predicted to impact the conformation of βB-βC loop, as G344 has torsion angles that are not
compatible with other amino acid residues.

81

a Bar graph showing total GluA1 lysate levels Co-IPed with WT SAP97 PDZ2, G344R, or G357S
normalized to the wild-type. Compared to wild-type SAP97 PDZ2, G344R and G357S have
significantly less interaction with GluA1 (bSAP97-G344R, n = 3 independent experiments, p =
Supplementary Figure 2.6: Supporting data for Figure 2.5
82
0.0008, two sample T-test; bSAP97-G357S, n = 3 independent experiments, p = 0.001, two sample
T-test). For (b) and (c), open circles are single pairs of control and transfected neurons, filled
circles represent the mean amplitudes (±SEM), insets show representative current traces from
control (black) and transfected (green) neurons with stimulation artifacts removed. Scale bars:
20ms, 20pA. Bar graphs show the average AMPAR-eEPSC amplitudes (±SEM) of DG granule
neurons of various experimental conditions. B Molecular replacement of GluA1 with GluA1-D7
occludes further augmentation of AMPAR-eEPSC amplitude produced by bSAP97-miR
expression in DG granule neurons. Average AMPAR-eEPSC amplitudes following expression of
GluA1 shRNA, GluA1-D7, and bSAP97-miR (green) in DG granule neurons (n = 5 pairs) is not
significantly different from the average AMPAR-eEPSC amplitudes produced by in GluA1-D7
molecular replacement alone (grey, n = 7 pairs; see Fig. 2.5f, g). p = 0.47, two sample T-test. c
Knocking down PSD-95, PSD-93, and SAP102 eliminates the synaptic augmentation produced by
bSAP97-miR expression in DG granule neurons. Average AMPAR-eEPSC amplitudes following
expression of the PSD-95, PSD-93, SAP102 triple MAGUK miR along with bSAP97-miR (green,
n = 6 pairs) is not significantly different from the average AMPAR-eEPSC amplitudes in the PSD-
95, PSD-93, SAP102 knockdown alone (grey, n = 7 pairs, see Fig. 2.3e, g). p = 0.56, two sample
T-test. d Imaging experiments showing dendritic spine localization of bSAP97 G344R-mCherry
(left) and bSAP97 G357S-mCherry (right) in GFP-filled DG granule neurons in entorhino-
hippocampal slices. E Molecular replacement of bSAP97 with bSAP97 -G344R in DG granule
neurons does not change surface AMPAR current amplitude. (Left) Representative current traces,
scale bar: 5s, 100pA. (Middle) Scatterplot where open circles are single pairs of control and
transfected neurons and the filled circle represents the mean amplitude (±SEM.) (Right) Bar graph
shows average surface AMPAR current amplitudes (±SEM) of control (black) and bSAP97-miR
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& bSAP97-G344R expressing (green) DG granule neurons (n = 5 pairs). p = 0.98, paired T-test. f
Coefficient of variation (CV) analysis of AMPAR-eEPSCs from pairs of control and bSAP97-miR
/ bSAP97-miR & bSAP97-G344R/ bSAP97-miR & bSAP97-G357S expressing DG granule
neurons. CV
-2
ratios are graphed against the mean amplitude ratio for each pair. Open circles (left):
green for bSAP97-miR, n = 8 pairs; red for bSAP97-miR & bSAP97-G344R, n = 7 pairs; blue for
bSAP97-miR & bSAP97-G357S, n = 7 pairs. Filled circles (right): mean ± SEM for each condition.
*p < 0.05; n.s., not significant. All statistical tests performed were two-tailed.
84
CHAPTER 3: AN OPTOGENETIC METHOD FOR INVESTIGATING
PRESYNAPTIC MOLECULAR REGULATION
3.1 Abstract

While efficient methods are well-established for studying postsynaptic protein regulation of
glutamatergic synapses in the mammalian central nervous system, similarly efficient methods are
lacking for studying proteins regulating presynaptic glutamatergic synapse function. In the present
study, we introduce an optical/electrophysiological method for investigating presynaptic
molecular regulation. Here, using an optogenetic approach, we selectively stimulate genetically
modified presynaptic CA3 pyramidal neurons in the hippocampus and measure optically induced
excitatory postsynaptic currents produced in unmodified postsynaptic CA1 pyramidal neurons.
While such use of optogenetics is not novel, previous implementation methods do not allow basic
quantification of the changes in synaptic strength produced by genetic manipulations. We find that
incorporating simultaneous recordings of fiber volley amplitude provides a control for optical
stimulation intensity and, as a result, creates a metric of synaptic efficacy that can be compared
across experimental conditions. In the present study, we utilize our new method to demonstrate
that inhibition of synaptotagmin 1 expression in CA3 neurons leads to a significant reduction in
Schaffer collateral synapse function, an effect that is masked with conventional electrical
stimulation. Our hope is that this method will expedite our understanding of molecular regulatory
pathways that govern presynaptic function.

85
3.2 Introduction

Highly specific, efficient and quantitative methods currently exist for studying the
influence of postsynaptic proteins on glutamatergic synapse function in the mammalian central
nervous system. For example, viral and biolistic approaches may be used to restrict genetic
manipulations to postsynaptic neurons and the consequences of such manipulations may be
measured by electrophysiological recordings from individual transduced/transfected postsynaptic
neurons during electrical stimulation of unmodified presynaptic neurons (Shipman and Nicoll,
2012, Sadybekov et al., 2017, Paskus et al., 2019, Tian et al., 2018, Rao et al., 2019). However,
comparable methods are sorely lacking for the study of proteins regulating presynaptic
glutamatergic synapse function. While viral methods may be used to restrict genetic manipulations
to presynaptic neurons, the consequences of such manipulations on synaptic function require
electrophysiological recordings from postsynaptic neurons. Even with high titer viruses, a mixed
population of transduced/untransduced presynaptic neurons are present. As a result, electrical
stimulation of presynaptic axons in this preparation produces neurotransmitter release from both
genetically modified and unmodified neurons which precludes an accurate measurement of the
impact the presynaptic genetic modification has on synaptic efficacy in electrophysiological
recordings from postsynaptic neurons.
At present, the only way to ensure genetic modification of all presynaptic neurons is to
engineer knockout/knockin mouse lines. However, traditional germline approaches result in the
genetic modification of both pre- and postsynaptic neurons often making it difficult to separate
potential pre- and postsynaptic roles for the proteins being studied. To limit genetic modification
to presynaptic neurons, conditional knockout/knockin mice must be engineered and then crossed
to specific driver lines. This approach can be extremely costly and time-consuming, especially
86
when paralogous proteins need to be considered.
Difficulty in studying the regulation of presynaptic function has motivated the recent
development of new methods to identify changes in presynaptic function. For example, optical
tools have now been developed that fluoresce upon binding to glutamate and may be expressed in
either pre- or postsynaptic neurons (Marvin et al., 2013). While certainly useful in potentially
detecting alterations in glutamate release, it can be difficult to determine the physiological
relevance of such changes (e.g. whether such changes ultimately produce measurable alterations
in the number of glutamate receptors that are activated postsynaptically). Recognizing our current
lack of an efficient and rigorous approach to study presynaptic protein function at native
mammalian glutamatergic synapses in the CNS, we describe in the present study an
optical/electrophysiological method for investigating presynaptic molecular regulation.
In this study, we use an optogenetic approach to selectively stimulate presynaptic CA3
neurons expressing an RNAi against our protein of interest and measure optically induced
excitatory postsynaptic currents (oEPSCs) produced in unmodified postsynaptic CA1 neurons
using a conventional whole-cell patch clamping technique. While such usage of optogenetics-
driven genetic manipulation in presynaptic studies is not novel (Jackman et al., 2016, Turecek and
Regehr, 2019), previous implementation methods do not allow basic quantification of the changes
in synaptic strength produced by the genetic manipulations. This is because in order to make
meaningful comparisons of synaptic strengths across experimental conditions, a quantitative
measurement of stimulus strength is required to ensure a comparable number of CA3 neurons is
stimulated in each condition. With our novel method, we resolve this problem by simultaneously
measuring fiber volleys (FVs) from CA3 axons while recording from CA1 neurons. FV amplitude
is determined by the number of CA3 neurons firing action potentials. Thus, we are able to derive
87
a metric of synaptic efficacy for each recording that can be compared across conditions by
obtaining a CA1-oEPSC/CA3-FV amplitude ratio.
To assess the accuracy of this approach we determined whether changes in oEPSC/FV
amplitude ratios match increases in glutamatergic synapse density. By comparing hippocampal
slices cultured from two different developmental timepoints during a period of robust
synaptogenesis, we observe a significant increase in dendritic spine density with increased animal
age. We find that our method accurately matches this change by comparing NMDAR-oEPSC/FV
amplitudes ratios between the two age groups. Additionally, the present study utilizes the new
method to study an important and readily studied presynaptic protein that has not previously been
studied at the CA3-CA1 synapse: synaptotagmin 1. Using our method, we find that there is a
significant reduction in CA1 current amplitude following Syt1 knockdown in CA3 neurons. This
phenotype is masked when CA3 axons are stimulated electrically, and the significant reduction is
only discernible using optical stimulation, demonstrating the critical need for incorporating
optogenetics in our approach. Thus, our method of presynaptic interrogation represents a simple,
cost-effective, and time-efficient approach that does not require generating new transgenic mouse
lines for every protein of interest.

88
3.3 Results

Method Setup

Our method of presynaptic molecular interrogation combines optogenetics, postsynaptic
whole cell patch clamping, and extracellular field recordings. The setup begins with the expression
of channelrhodopsin in a population of presynaptic neurons. Here, to study the CA3-CA1 synapse,
we injected an AAV co-expressing channelrhodopsin (ChR2/H134R) and mCherry directly into
the CA3 region of cultured rat hippocampal slices. We verified successful viral transduction via
mCherry epifluorescence in the CA3 region and particularly in CA3 axons (Schaffer collaterals)
(Fig. 3.1a). We also biolistically transfected a CA1 neuron with tdTomato in order to visually
demonstrate the hippocampal slice setup where the CA3 axons expressing ChR2-mCherry overlap
with dendrites of CA1 pyramidal neurons (Fig. 3.1a, b). CA3 axons expressing ChR2 were
optically stimulated to evoke an AMPA receptor (AMPAR)-mediated oEPSC in CA1 pyramidal
neurons. Whole-cell recordings of a CA1 pyramidal neuron were made simultaneously with
extracellular field recordings from Schaffer collaterals (Fig. 3.1c). By aligning the optically
induced extracellular field recording trace with the AMPAR-oEPSC, we verified that the
postsynaptic responses (fEPSP and oEPSC) were aligned in time, and that we could isolate the
presynaptic fiber volley (Fig. 3.1d). Furthermore, we found that with increased optical stimulation
strength, there was a corresponding increase in both presynaptic fiber volley amplitude and
AMPAR-oEPSC amplitude, demonstrating that these measurements scale with light intensity (Fig.
3.1e). Fiber volley amplitude represents the number of CA3 pyramidal neurons firing action
potentials and, as a result, can be used as a readout of Schaffer collateral stimulation strength. To
create a metric of synaptic efficacy, CA1-oEPSC amplitude was then divided by fiber volley
89
amplitude to generate a normalized CA1-oEPSC/CA3-FV amplitude ratio that provides the
amount of postsynaptic current produced in a neuron by a given amount of presynaptic stimulation.

Method Validation 1 – Increased NMDAR-oEPSC/Fiber Volley amplitude ratio mirrors
glutamatergic synaptogenesis

Before commencing with presynaptic genetic manipulations, we first assessed whether our
method accurately reflects changes in glutamatergic neurotransmission. To this end, super-
resolution images of dendritic spines were obtained via Structured Illumination Microscopy, which
revealed that there is a 2.5-fold increase in CA1 pyramidal neuron dendritic spine density in
hippocampal slice cultures prepared from postnatal day 8 (P8) pups vs. postnatal day 6 (P6) pups
(Fig. 3.2a). This change in spine number reflects the high level of synaptogenesis occurring in the
brain during this time in postnatal development.
The vast majority of glutamatergic synapses are located on dendritic spines(Bourne and
Harris, 2008, Gray, 1959). Given that glutamatergic synapses can be silent (i.e. lacking AMPARs
but expressing NMDARs) (Kerchner and Nicoll, 2008) and that presynaptic release probability
does not change between P6 and P8 slices based on paired-pulse ratios (Fig. 3.2b), we reasoned
that by using our new method we would resolve a fold change in NMDAR-mediated currents in
P8 vs. P6 slices that was similar to the change we observed in dendritic spine density. We first
examined the oEPSC’s and the FV amplitudes over a range of light intensities to ensure that, with
increasing light intensity, the FV amplitudes and oEPSCs did not change independently of one
another. The collected range data consisted of at least 3 data points per cell. We plotted the
oEPSC’s as a function of FV amplitudes and fit the measurements from each cell with a linear
regression line. Each linear fit represents a recording from a single neuron. In every recording, we
90
observed a linear relationship between oEPSC and FV amplitudes (Fig. 3.2c, e). We then averaged
the NMDAR-oEPSC/FV ratios in each condition and, remarkably, resolved a 2.5-fold increase in
NMDAR-oEPSC/fiber volley amplitude ratio in the P8 slices compared to the P6 slices (Fig. 3.2e),
a near perfect match to the increase in dendritic spine number we observed (Fig. 3.2a). Furthermore,
we assessed whether a single measurement of the NMDAR-oESPC/FV ratio at maximum optical
stimulation (Max NMDAR-oEPSC/FV ratio) was sufficient to maintain the accuracy. For each of
the recordings plotted in Figure 2C, a single measurement of NMDAR-oEPSC and FV amplitude
was taken for each neuron at maximum optical stimulation and plotted as individual circles in
Figure 2D. We also took Max NMDAR-oEPSC/FV ratios from additional neurons where fewer
than 3 data points were collected. These Max NMDAR-oEPSC/FV ratios were averaged for each
condition, which resulted in a 2.4-fold increase in P8 slices, comparable to the previous
measurements (Fig. 3.2d, e). Such data demonstrate that our method only requires a single
oEPSC/FV amplitude ratio per neuron to maintain a high level of accuracy. Using the same
approach, we also compared AMPAR-oEPSC/FV ratios in P6 and P8 slices and found a 2.1-fold
increase in P8 slices compared to the P6 slices (Fig. 3.2f, h). Averaging only the Max AMPAR-
oEPSC/FV ratios from each cell instead of taking multiple measurements resulted in a 2.2-fold
change (Fig. 3.2g), again demonstrating that a single ratio measurement is sufficient for
maintaining our method’s accuracy. The smaller fold increase we observe in AMPAR-oEPSC/FV
ratio with respect to that of NMDARs is likely explained by an increased number of silent synapses
in slices from P8 versus P6 animals. Taken together, the functional NMDAR-oEPSC/FV ratio data
from our method were nearly identical to that of dendritic spine analysis and therefore demonstrate
that our technique represents a highly accurate method of measuring the strength of synaptic
contact between neurons.
91
Method Validation 2 – Synaptotagmin 1 Knockdown
Next, to establish that this method allows for a new and accurate approach to studying
presynaptic genetic manipulations, we examined the effects of knocking down (KD)
synaptotagmin 1 (Syt1) in the CA3 pyramidal neurons. Syt1 is a well-established calcium sensor
on presynaptic vesicles, involved in synchronous vesicular neurotransmitter release and rapid
synaptic transmission (Geppert et al., 1994). The role of presynaptic Syt1 in glutamatergic
neurotransmission has been examined in dissociated cortical cultures (Maximov and Sudhof, 2005,
Xu et al., 2007), hippocampal cultures (Bacaj et al., 2013), dentate granule cell-basket cell
synapses (Kerr et al., 2008), and CA1-Subiculum synapses (Xu et al., 2012, Bacaj et al., 2013).
Surprisingly, the role of Syt1 at the CA3-CA1 synapse, one of the most studied synapses in the
brain, has not yet been explored. Using an AAV co-expressing channelrhodopsin and Syt1 shRNA,
we knocked down Syt1 expression in CA3 pyramidal neurons. An AAV expressing only
channelrhodopsin (control), as well as an AAV co-expressing channelrhodopsin and a scrambled
shRNA, were each injected into the CA3 region of the two control groups. The Syt1 shRNA was
previously validated (Xu et al., 2012) and we verified via western blot that it significantly reduced
Syt1 levels in HEK293 cells compared to a scrambled shRNA (Fig. 3.3a). To test if the
optogenetics component was necessary, as well as whether electrical stimulation of Schaffer
collaterals would be sufficient to detect an alteration in synaptic efficacy with Syt1 KD, we first
electrically stimulated Schaffer collaterals (Fig. 3.3b), and found no change in either Max
AMPAR- or Max NMDAR-electrically evoked excitatory post synaptic current/fiber volley
(AMPAR- or NMDAR-eEPSC/FV) amplitude ratios between Syt1 KD and control groups (Fig.
3.3c, d). We speculated that the indiscriminate electrical stimulation of Schaffer collaterals from
both virally-transduced Syt1 KD and untransduced CA3 neurons may have masked a change in
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synaptic transmission.
Using the same experimental setup, we then optically stimulated the Schaffer collaterals in
order to selectively stimulate CA3 neurons co-expressing channelrhodopsin and the Syt1 shRNA
(Fig. 3.4a). In marked contrast to electrical stimulation, optical stimulation revealed a 60%
reduction in the Max AMPAR-oEPSC/FV ratio and a 55% reduction in the Max NMDAR-
oEPSC/FV ratio in the Syt1 KD group compared to both the control group and the scrambled
shRNA group (Fig. 3.4b, c). Comparable ranges of optical axonal stimulation were used in all
conditions as shown by similar average FV amplitudes (Fig. 3.4b, c). The similar reductions we
observe in Max AMPAR- and NMDAR-oEPSC/FV ratios are consistent with reduced Syt1
expression in CA3 pyramidal neurons inhibiting evoked glutamate release onto both glutamate
receptor subtypes. We also examined whether asynchronous release was affected by knocking
down Syt1 in CA3 pyramidal neurons. It is generally believed that Syt1 plays a selective role in
fast, synchronous release, and that removing Syt1 does not interfere with normal asynchronous
release (Maximov and Sudhof, 2005, Xu et al., 2012). As expected, we found no significant
differences in the frequency or amplitude of asynchronous release events with Syt1 knockdown
compared to the control group (Fig. 3.4d). Altogether, our results demonstrate that Syt1 plays a
significant role in synchronous glutamate release at CA3-CA1 synapses. Furthermore, our side-
by-side experiments comparing electrical vs. optical stimulation in the same setup demonstrate the
utility of our new method, given that electrical stimulation failed to resolve a change in glutamate
release following CA3 pyramidal neuron transduction with the Syt1 RNAi.

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3.4 Discussion

Historically, the ease of studying the contribution of presynaptic proteins to synaptic
regulation has lagged behind the study of synaptic regulation by postsynaptic proteins. Isolation
of genetic manipulations to specific populations of presynaptic neurons often comes with great
cost, and precise methods of quantifying the effects these manipulations have on synaptic efficacy
are sorely lacking. Isolating the presynaptic function of a protein in glutamatergic synapses has
thus far been predominantly limited to two common approaches: dissociated neurons in culture
and transgenic mouse lines. Using dissociated neurons is a straightforward system for making
genetic manipulations, but its critical limitation stems from the fact that cultured neurons do not
maintain their original, endogenous synaptic circuitry. Instead, cultured neurons form
indiscriminate synaptic connections regardless of their endogenous connections or cell type. The
hippocampus, for example, has discrete regions of neuronal subtypes such as CA3 and CA1
pyramidal neurons and dentate granule neurons, and it has been increasingly appreciated that there
is pathway-specific regulation that functionally differentiates these synapses (Rao et al., 2019,
Siddiqui et al., 2013, Farris et al., 2019, Alkadhi, 2019, Zhu et al., 2018). Our novel approach
allows for keeping the endogenous circuitry of synapses intact by utilizing hippocampal slices. In
cultured hippocampal slice preparations, genetic manipulations can easily be targeted to the CA3
subregion, and whole-cell recordings from CA1 pyramidal neurons allow the experimenter to
assess the consequences of the presynaptic manipulation at CA3-CA1 synapses with a high level
of confidence.
Currently the most sophisticated approach for studying presynaptic regulation requires
generating knockout mouse lines. Generation of such mouse lines is not a trivial process and can
come at great cost both in terms of both money and time. However, one potential problem with
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using this approach is that global knockout models limit the ability to discern whether any observed
phenotype is due to a pre- or postsynaptic role of the protein. In order to conclusively identify the
presynaptic function of a protein in transgenic mice, the genetic manipulation must be limited to a
specific population of presynaptic neurons. For example, to study the presynaptic function of
synaptotagmin 1 using transgenic mice in CA3-CA1 synapses, the genetic manipulation must be
limited to CA3 pyramidal neurons. This isolated manipulation is especially important given that
canonical presynaptic proteins such as synaptotagmins have also been shown to play postsynaptic
roles in synaptic regulation (Hussain et al., 2017). Furthermore, germline knockout models can be
lethal, and even if the mouse reaches viability, if the protein is absent throughout development,
this can lead to compensatory mechanisms and downstream effects that hinder our ability to isolate
the specific role the protein plays in the synapse. Our approach not only allows for a more efficient
way of selectively manipulating presynaptic neurons genetically, but also allows the observation
of the immediate consequences of the genetic manipulation which reduces the likelihood of
compensatory mechanisms occluding changes in phenotype.
In the present study we validated our novel method by accurately matching results from
dendritic spine analysis comparing cultured hippocampal slices from P6 and P8 rat pups. During
this period of rapid synaptogenesis, there was a significant increase in spine density, which our
metric mirrored remarkably well. We then utilized the method to study the presynaptic role of Syt1
in the CA3-CA1 synapse for the first time. Based on previous literature, we speculated that
knocking down expression of Syt1 in CA3 neurons would disrupt transmission in the CA3-CA1
synapses, and indeed we found a significant reduction in the oEPSC/FV ratios for both AMPAR-
and NMDAR-mediated currents. A complete absence of synchronous release, however, has been
reported in Syt1 knockout models at other synapses. The remaining synchronous release we
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observe at CA3-CA1 synapses following Syt1 knockdown may be supported by either residual
Syt1 protein or additional synaptotagmin-related proteins. While we used an RNAi to influence
protein expression in the present study, it is important to note that our method can be used with
other forms of genetic modification as well that lead to complete elimination of protein expression
(e.g. CRISPR-based strategies, CRE/lox mouse lines, etc.). In our study we found a similar level
of reduction in both AMPAR- and NMDAR-mediated currents following presynaptic knockdown
of Syt1, which would be expected from a synapse where glutamate release is inhibited. Consistent
with previous reports, we also find no change in asynchronous release properties with reduced
Syt1 expression. In future experiments using our new method, it will be interesting to explore the
roles of other synaptotagmin isoforms at CA3-CA1 synapses.
A time- and cost-effective approach to rigorously study the molecular regulation of
presynaptic function within a native mammalian synapse had yet to exist prior to this present study.
Our hope is that this novel approach will lead to rapid advancements in understanding molecular
regulation of presynapse function. Furthermore, the utility of our method can be expanded by
incorporating biolistic transfection of postsynaptic neurons for simultaneous pre- and postsynaptic
genetic control (Fig. 3.1a). By genetically controlling each side of the synapse independently, this
combination of methods will open doors for a new line of synaptic studies such as investigating
the roles of specific isoform combinations of transsynaptic adhesion molecules. Transsynaptic
adhesion molecules regulate synapse development, as well as play a role in synaptic transmission
and plasticity (Jang et al., 2017). In the hippocampus, postsynaptic dendritic spines with larger
PSDs and higher surface AMPAR expression have corresponding presynaptic terminals with
larger active zones and more docked vesicles. Such findings demonstrate the importance of pre-
and postsynaptic coordination, most likely mediated by trans-synaptic adhesion molecules (Jang
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et al., 2017, Rudenko, 2017, Kilinc, 2018). These proteins, which include cadherins, ephrins,
neuroligins, and neurexins, are especially important to investigate given that many have been
strongly implicated in cognitive disorders such as autism and schizophrenia (Sudhof, 2008, Zhang
et al., 2018, Reichelt et al., 2012, Guang et al., 2018). However, they are difficult to efficiently
study because they have multiple isoforms, with neurexin alone having thousands of potential
splice variants (Ullrich et al., 1995, Treutlein et al., 2014). Our new method can circumvent
existing methods’ limitations, expedite the exploration of these proteins, and ultimately lead to
enhancing our understanding of the synapse.

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3.5 Materials and Methods

Experimental constructs

pAAV.CAG.hChR2(H134R)-mCherry.WPRE.SV40 and the AAV9 viral particles produced from
the plasmid were a gift from Karl Deisseroth (Addgene viral prep #100054-AAV9). Syt1 shRNA
target sequence 5’-GAGCAAATCCAGAAAGTGCAA-3’ was previously determined and
validated (Xu et al., 2012). pAAV.H1.Syt1shRNA.CAG.hChR2(H134R)-mCherry was
constructed and packaged by VectorBuilder by modifying the original hChR2 pAAV plasmid to
include the Syt1 shRNA behind an H1 promoter (Vector ID VB170324-1065bbv).

pAAV.H1.scrambledshRNA.CAG.hChR2(H134R)-mCherry was also constructed and packaged
by VectorBuilder by modifying the Syt1 shRNA AAV plasmid to instead express a scrambled
shRNA (Vector ID VB191113-1716anp). Rat synaptotagmin 1 in pcDNA3.1
+
/C-(K)DYK was
acquired from GenScript (NM_001033680.2). pFUGW-GFP was used to identify transfected
neurons in spine density analysis.

Slice virus injection and electrophysiology

All experimental procedures were carried out in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals and approved by the University of
Southern California Institutional Animal Care and Use Committee. This study was carried out in
compliance with the ARRIVE guidelines. 400 µm rat organotypic hippocampal slice cultures were
prepared from both male and female P6 to P8 Sprague Dawley rats as previously described
(Bonnici and Kapfhammer, 2009, Prang et al., 2001, Stoppini et al., 1991). AAV9 viral particles
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from pAAV plasmids expressing channelrhodopsin (ChR2(H134R) + mCherry only, Syt1 shRNA
+ ChR2(H134R) + mCherry, or scramble shRNA + ChR2(H134R) + mCherry) were injected into
the CA3 pyramidal layer of the organotypic hippocampal slice cultures on DIV1 using a Nanoject
II device (Drummond Scientific). Successful viral transduction was later verified by mCherry
epifluorescence in the CA3 region and particularly in the Schaffer collaterals (Fig. 3.1a). Culture
media was exchanged every other day until recording on DIV12-13. During recordings, slices were
maintained in room-temperature artificial cerebrospinal fluid (aCSF) external solution containing
(in mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose, 4 CaCl2, and 4 MgSO4. 5
µM 2-chloroadenosine and 0.1mM picrotoxin were also added to the aCSF to dampen epileptiform
activity and block GABAA receptor activity, respectively. Osmolarity was adjusted to 310-315
mOsm. aCSF was saturated with 95% O2/5% CO2 throughout recording. Borosilicate extracellular
field recording electrodes were filled with the same aCSF external solution. Borosilicate whole
cell recording electrodes were filled with an internal, whole-cell recording solution containing (in
mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.3 EGTA, 5 QX-314, 4 Mg-ATP, and 0.3 Na-GTP.
The internal solution was adjusted to pH 7.3-7.4 and osmolarity of 290-295 mOsm.

Virally transduced CA3 axons were either electrically stimulated with a monopolar glass electrode
filled with aCSF or optically stimulated with blue light to evoke a postsynaptic response in CA1
pyramidal neurons. Whole-cell recordings of a CA1 pyramidal neuron’s AMPAR- and NMDAR-
mediated current amplitudes were made simultaneously with extracellular field recordings in the
Schaffer collaterals (Fig. 3.1c). Synaptic responses were acquired using a Multiclamp 700B
amplifier (Molecular Devices). AMPAR-mediated EPSCs were measured at -70mV. NMDAR-
mediated EPSCs were measured at +40mV, temporally isolated from AMPAR currents by
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measuring amplitudes 250ms after stimulus onset. In most cases, AMPAR- and NMDAR-
mediated currents were recorded from the same neuron by changing the membrane potential.
Optically induced extracellular field recording trace was merged with the postsynaptic whole cell
recording trace to verify that the postsynaptic responses (EPSP and EPSC) were aligned in time
and also to isolate the presynaptic fiber volley (Fig. 3.1d). It was demonstrated that with increased
optical stimulation strength, there was a corresponding increase in both presynaptic fiber volley
amplitude and postsynaptic current amplitude (Fig. 3.1e). No more than one simultaneous
recording was performed on any given hippocampal slice. To assess asynchronous release,
asynchronous EPSCs with an amplitude of ≥5 pA and a rate of rise of ≥4pA/ms were automatically
detected and analyzed with customized IGOR software (Herring et al., 2013).

Immunoblotting

To validate the Syt1 shRNA construct, HEK293 cells were co-transfected with a rat Syt1 construct
and pAAV.H1.Syt1shRNA.CAG.hChR2(H134R)-mCherry using Lipofectamine 2000. Syt1 co-
transfected with pAAV.H1.ScrambleshRNA.CAG.hChR2(H134R)-mCherry was used as the
control. Lysates were prepared 72 hours post transfection and lysed in RIPA buffer containing
protease inhibitor mix (Thermo Scientific, Halt Protease Inhibitor Cocktail). Proteins were
resolved by SDS-PAGE and analyzed by western blot with antibodies against rat synaptotagmin 1
(1:1000, Synaptic Systems Cat#105 011) and b-actin (1:1000, Cell Signaling Technology
Cat#4970). Secondary antibodies used were anti-mouse and anti-rabbit IgG, HRP-linked
antibodies, respectively (1:10000, Cell Signaling Technology Cat#7076/7074).

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Spine density analysis

CA1 pyramidal neurons in cultured hippocampal slices prepared from P6 and P8 rat pups were
biolistically transfected with a pFUGW-GFP construct on DIV1. On DIV7, slices were fixed in
4%PFA/4% sucrose in PBS, washed 3 times in PBS, and cleared using an abbreviated SeeDB-
based protocol (Ke et al., 2013). Images were acquired using super-resolution microscopy (Elyra
Microscope System, Zeiss), blinded to condition, with an oil-immersion 100x objective lens.
Image acquisition and analysis were carried out as described previously (Tian et al., 2018,
Sadybekov et al., 2017).

Statistical analysis

Electrophysiological recordings of normalized EPSC amplitudes/FV amplitudes, paired pulse
facilitation, and asynchronous release were analyzed using a Wilcoxon Rank Sum Test. Data
analysis was performed using Igor Pro (Wavemetrics). Spine density data was analyzed using
unpaired Student’s t-test. P-value of < 0.05 was considered statistically significant. Error bars
represent standard error of the mean measurement. Sample sizes in the present study are similar to
those reported in the literature (Incontro et al., 2018, Herring and Nicoll, 2016).

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a Image of rat hippocampal slice culture setup for recording CA1 pyramidal neuron currents with
optical stimulation of Schaffer collaterals. The CA3 region was injected with an AAV expressing
ChR2 tagged with mCherry, and mCherry fluorescence can be seen in both the CA3 pyramidal
neuron cell bodies and axons (Schaffer Collaterals). A biolistically transfected CA1 pyramidal
neuron is also visible by its tdTomato fluorescence. b Schematic illustration of a CA3-CA1
Figure 3.1: Method setup

Figure 3.2: Increased NMDAR-oEPSC/Fiber Volley amplitude ratio mirrors glutamatergic
synaptogenesisFigure 3.3: Method setup

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synapse in our setup. ChR2-mCherry is expressed in the presynaptic CA3 axons via AAV virus,
and biolistically-transfected tdTomato is optionally expressed postsynaptically in the CA1 neuron.
c Schematic illustration of experimental setup. Virally transduced CA3 pyramidal neurons
expressing ChR2 are stimulated optically using 470 nm blue light to evoke a postsynaptic response
in CA1 pyramidal neurons. Whole-cell recordings of a CA1 neuron were made simultaneously
with extracellular field recordings in the Schaffer Collaterals. d Optically induced extracellular
field recording trace merged with postsynaptic whole cell recording trace, verifying that the two
traces align in time and illustrating the presynaptic fiber volley. Scale bar: 20ms. e Demonstration
of control over optical stimulation strength and the corresponding increase in both presynaptic
fiber volley amplitude and postsynaptic current amplitude, evidence that these measurements scale
with light stimulus intensity. Scale bar: 20ms.

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a Hippocampal slice cultures from P8 rats have increased spine density compared to those from
P6 rats. (Left) Representative CA1 dendritic spine images in P6 vs P8 slices, neurons transfected
with GFP. (Right) Bar graph showing averaged spine density in P6 vs P8 slices normalized to P6
slices (P6: 0.12 ± 0.019 spines/µM, n = 11, P8: 0.29 ± 0.039 spines/µM, n = 14, p = 0.001,
Student’s t test). For (b)-(h), Wilcoxon rank sum test was used; *p < 0.05. b Representative traces
(left) and mean ± SEM paired-pulse facilitation ratios for P6 and P8 CA1 neurons (right) (P6: n =
11; P8: n = 7, p = 1, n.s., not significant). c NMDAR-oEPSC’s plotted as a function of fiber volley
amplitude over a range of stimulation strengths (range = 3+ data points acquired per cell);
measurements from each cell were fitted with a linear regression line. Inset representative traces
demonstrate increasing fiber volley amplitude and increasing NMDAR-oEPSC amplitude. d Max
NMDAR-oEPSC/FV amplitude measurements, taken at maximum optical stimulation, for each
Figure 3.2: Increased NMDAR-oEPSC/Fiber Volley amplitude ratio mirrors glutamatergic
synaptogenesis

Figure 3.4: Deficits in CA3-CA1 excitatory synaptic transmission following Syt1 knockdown
in CA3 pyramidal neurons are not observed with electrical stimulationFigure 3.5: Increased
NMDAR-oEPSC/Fiber Volley amplitude ratio mirrors glutamatergic synaptogenesis

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cell in P6 and P8 slices. Each point represents the Max NMDAR-oEPSC/fiber volley amplitude
of one cell. Measurements from each group (P6 vs P8) were fitted with a linear regression line. e
(Left) Averaged and normalized NMDAR-oEPSC/FV amplitude ratio in P6 vs. P8 slices (P6: n =
7, P8: n = 8, p = 0.02). (Right) Averaged and normalized Max NMDAR-oEPSC/FV ratio in P6 vs.
P8 slices (P6: n = 10, P8: n = 13, p = 0.035). The red dotted line shows fold change in dendritic
spine density in (A). f AMPAR-oEPSC’s plotted as a function of fiber volley amplitude over a
range of stimulation strengths (range = 3+ data points acquired per cell). Measurements from each
cell were fitted with a linear regression line. Inset representative traces demonstrate increasing
fiber volley amplitude and increasing AMPAR-oEPSC amplitude. g Max AMPAR-oEPSC/FV
amplitude measurements, taken at maximum optical stimulation, for each cell in P6 and P8 slices.
Each point represents the Max AMPAR-oEPSC/fiber volley amplitude ratio of one cell.
Measurements from each group (P6 vs P8) were fitted with a linear regression line. h (Left)
Averaged and normalized AMPAR-oEPSC/FV amplitude ratio in P6 vs. P8 slices (P6: n = 10, P8:
n = 11). (Right) Averaged and normalized Max AMPAR-oEPSC/FV ratio in P6 vs. P8 slices (P6:
n = 21, P8: n = 19, p = 0.0005)

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a Western blot showing knockdown of Syt1 with Syt1 shRNA in HEK293 cells. Top: probed for
Syt1, bottom: probed for ß-Actin. The same blot was used to probe for both proteins. Images have
been cropped for clarity. Full-length blots are presented in Supplementary Figure 1. b Schematic
illustration of recording setup using electric stimulation. AAV expressing either ChR2 only
(control) or ChR2 with Syt1 shRNA were injected into CA3 neurons, and CA3 axons were
Figure 3.3: Deficits in CA3-CA1 excitatory synaptic transmission following Syt1 knockdown
in CA3 pyramidal neurons are not observed with electrical stimulation

Figure 3.6: Syt1 knockdown diminishes CA3-CA1 excitatory synaptic transmission with
optical stimulation of CA3 pyramidal neuronsFigure 3.7: Deficits in CA3-CA1 excitatory
synaptic transmission following Syt1 knockdown in CA3 pyramidal neurons are not
observed with electrical stimulation

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electrically stimulated with an electrode. Schaffer collateral field recordings and whole cell patch
recordings of CA1 pyramidal neurons were simultaneously acquired. In (c)&(d), Wilcoxon rank
sum test was used. *p < 0.05, n.s., not significant. c (Left) Max AMPAR-eEPSC/FV ratios per
cell in control vs Syt1 shRNA groups following electric stimulation. Each point represents the
Max AMPAR-eEPSC/FV amplitude ratio of one cell; measurements from each group were fitted
with a linear regression line. Insets show representative traces from control (black) and Syt1
shRNA (gray) with stimulation artifacts removed. (Center) Averaged and normalized Max
AMPAR-eEPSC/FV amplitude ratio in control vs. Syt1 shRNA groups following electric
stimulation (control n = 14, Syt1 shRNA n = 14, p = 0.31). (Right) Averaged fiber volley
amplitudes in control vs. Syt1 shRNA groups (control n = 14, Syt1 shRNA n = 14, p = 0.51). d
(Left) Max NMDAR-eEPSC/FV ratios per cell in control vs Syt1 shRNA groups following electric
stimulation. Each point represents the Max NMDAR-eEPSC/fiber volley amplitude ratio of one
cell; measurements from each group were fitted with a linear regression line. Insets show
representative traces from control (black) and Syt1 shRNA (gray) with stimulation artifacts
removed. (Center) Averaged and normalized Max NMDAR-eEPSC/FV amplitude ratio in control
vs. Syt1 shRNA groups following electric stimulation (control n = 12, Syt1 shRNA n = 11, p =
0.12). (Right) Averaged fiber volley amplitudes in control vs. Syt1 shRNA groups (control n = 12,
Syt1 shRNA n = 11, p = 0.79).

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a Schematic illustration of recording setup using optogenetic stimulation. AAV expressing: (1)
ChR2 only (control), (2) ChR2 with scrambled shRNA (scrambled shRNA), or (3) ChR2 with Syt1
shRNA were injected into the CA3 region of hippocampal slices. CA3 pyramidal neuron axons
were optically stimulated with blue light. Schaffer collateral field recordings and whole cell patch
recordings of CA1 pyramidal neurons were simultaneously acquired. For (b)-(d), Wilcoxon rank
sum test was used. *p < 0.05, n.s., not significant. b (Left) Max AMPAR oEPSC/FV ratios per cell
in control, scrambled shRNA & Syt1 shRNA groups following optical stimulation. Each point
represents the maximum AMPAR-oEPSC/FV amplitude ratio of one cell; measurements from
each group were fitted with a linear regression line. Insets show representative traces from control
Figure 3.4: Syt1 knockdown diminishes CA3-CA1 excitatory synaptic transmission with
optical stimulation of CA3 pyramidal neurons

Supplementary Figure 2.7: bSAP97 immunolabeling in the hippocampus is specific and
overlaps with MAP2 in DG granule neurons.Figure 3.8: Syt1 knockdown diminishes CA3-
CA1 excitatory synaptic transmission with optical stimulation of CA3 pyramidal neurons

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(black) and Syt1 shRNA (red). (Center) Averaged and normalized Max AMPAR-oEPSC/FV
amplitude ratio in control, scrambled shRNA & Syt1 shRNA groups (n = 6, 8, 7 respectively,
control vs. Syt1 shRNA p = 0.007, scrambled vs. Syt1 shRNA p = 0.0037). (Right) Averaged fiber
volley amplitudes in control, scrambled shRNA & Syt1 shRNA groups (control n = 6, scrambled
shRNA n = 8, Syt1 shRNA n = 7, control vs. Syt1 shRNA p = 0.63, scrambled vs. Syt1 shRNA p
= 0.46). c (Left) Maximum NMDAR-oEPSC/FV ratios per cell in control, scrambled shRNA &
Syt1 shRNA groups. Insets show representative traces from control (black) and Syt1 shRNA (red).
(Center) Averaged and normalized NMDAR-oEPSC/FV amplitude ratio in control, scrambled
shRNA & Syt1 shRNA groups (n = 8, 7, 7 respectively, control vs. Syt1 shRNA p = 0.022,
scrambled vs. Syt1 shRNA p = 0.0023). (Right) Averaged fiber volley amplitudes in control,
scrambled shRNA & Syt1 shRNA groups (n = 8, 7, 7 respectively, control vs. Syt1 shRNA p =
0.46, scrambled vs. Syt1 shRNA p = 0.12). d Asynchronous release following optically induced
synchronous release in control (black) vs. Syt1 shRNA (red) groups. (Left, Top) Representative
traces, 10 sweeps of release events merged to show individual release events. (Left, Bottom)
Proportion of release events over one second following initial optical stimulus. (Right) Frequency
and amplitudes of asynchronous release events in control vs. Syt1 shRNA groups (n = 6 each group,
both frequency and amplitude, p = 0.9372 and 0.4848 respectively).

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CHAPTER 4: CONCLUSION

Dysfunction in synaptic transmission is believed to play a significant role in the
pathophysiology of various complex brain disorders. However, the definitive pathogenic
mechanisms underlying disorders such as schizophrenia remain unclear despite decades of
research. In order to uncover these mechanisms, we must first expand our understanding of the
basic regulatory and functional mechanisms of synaptic transmission. To this end, the work in this
dissertation delved into the synapse-specific regulatory roles of proteins implicated in brain
disorders.
Chapter 2 described the discovery of the synaptic role of SAP97, a MAGUK protein that
had been largely disregarded in synapse literature due to a myriad of previous studies finding no
synaptic role for SAP97. Driven by the knowledge that SAP97 has been strongly implicated in a
synaptic disorder, we employed immunohistochemistry and visually identified the dentate gyrus
of the hippocampus as where SAP97 is endogenously expressed and likely to play a synaptic
regulatory role. Through thorough characterization, we found that SAP97 plays a critical synaptic
regulatory role in the dentate gyrus but not in the CA1 region, where it had been previously studied
despite little endogenous expression in this region. We also determined that the mechanism behind
SAP97’s synaptic role is likely via direct binding to GluA1’s PDZ-binding domain in its C-tail,
which was also previously regarded as having no synaptic role when studied in the CA1 region. In
discovering this mechanism, we have identified synaptic roles for both SAP97 and GluA1’s C-tail
for the first time.
The primary reason for why these roles had remained undiscovered is that their roles are
likely specific to the perforant pathway-dentate gyrus synapse. It remains to be elucidated why
SAP97 seems to play a critical regulatory role specifically in the dentate gyrus but not in the CA1
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region, as well as why SAP97, despite the similarities, seems to play the opposite role to the other
traditional MAGUKs such as PSD-95, PSD-93, and SAP102. One hypothesis for what
differentiates SAP97 from the other MAGUKs is that the canonical form of SAP97 in the brain
expresses the L27 domain in the N-terminus, whereas the other MAGUKs instead express a
palmitoylation motif in this region. The L27 domain may be responsible for the perisynaptic
localization of SAP97, while the other MAGUKs localize in the PSD. SAP97 may be holding
AMPARs in the perisynaptic regions through the direct interaction between SAP97 and GluA1’s
PDZ-binding C-tail. When this interaction between the two proteins is disrupted, either by SAP97
knockdown or by schizophrenia-related mutations in SAP97, AMPARs are released and grabbed
by the other MAGUKs in the PSD, thereby increasing the synaptic expression of AMPARs and
consequently leading to increased AMPAR-mediated transmission.
It should be noted that while SAP97 expression seemed to be highly concentrated in the
dendrites of DG granule neurons, SAP97 expression was also observed in the nuclei of CA1
pyramidal neurons. Dendritic SAP97 expression was not visible in CA1 pyramidal neurons, which
likely explains why knocking down SAP97 in these neurons did not lead to any changes in synaptic
transmission. What causes the different expression patterns of SAP97 in these two regions, and
the consequent distinct synaptic roles of SAP97 in each region, remains a question. A hypothesis
for why the pattern of SAP97 expression varies across these regions and why SAP97 plays distinct
roles in these regions is that particular isoforms of SAP97 may be expressed in each region. As
described previously, there are many alternatively spliced isoforms of SAP97. The inserts I3 and
I2 in the alternatively spliced region between the SH3 and GUK domains are of particular interest
because these inserts may determine where SAP97 localizes. Isoforms expressing both I3 and I2
are truncated early and are likely to be not functional (McLaughlin et al., 2002). The I3-containing
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isoforms were found to localize at sites of cell-cell contact, while the I2-containing isoforms
localized within the nucleus in epithelial cells (McLaughlin et al., 2002, Mori et al., 1998), which
could explain the localization patterns we saw in the hippocampus.
What drives this insert-dependent differential localization may be through SAP97’s
interaction with protein 4.1N via the I3 insert. In neurons, protein 4.1N plays a critical structural
role by stabilizing spectrin-actin interactions. Its homolog Protein 4.1R is responsible for recruiting
SAP97 to membranes in erythrocytes (Hanada et al., 2003). The I2 insert has been found to not
bind to protein 4.1N, while removing the I3 insert from SAP97 led to a dramatic loss in spine
localization of SAP97 (Rumbaugh et al., 2003, Lue et al., 1994). Intriguingly, evidence from our
lab and previous literature both suggest that protein 4.1N is also highly concentrated in the dentate
gyrus but not in the CA1 region of the hippocampus. Further, preliminary immunohistochemistry
experiments from our lab staining for specifically the I3 insert shows high dendritic expression of
SAP97 in DG granule neurons and not in the CA1 pyramidal neurons, whereas staining for the I2
insert shows SAP97 expression in the nuclei in both the DG granule neurons and CA1 pyramidal
neurons. In addition to these promising preliminary findings, it is also noteworthy that GluA1’s
C-tail contains a protein 4.1N binding domain (Shen et al., 2000). It is likely that the interactions
amongst SAP97, GluA1’s C-tail, and protein 4.1N underly the unique, dentate gyrus-specific
mechanism discussed in Chapter 2. Further studies on characterizing the role of protein 4.1N in
the dentate gyrus as well as how it fits into the mechanism with SAP97 and GluA1 will be critical
in understanding the unique nature of dentate gyral synaptic transmission.
Given that the dentate gyrus has been strongly implicated in schizophrenia (Yun et al.,
2016, Das et al., 2014, Tamminga et al., 2010, Kawano et al., 2015, Ota et al., 2017, Falkai et al.,
2016, Kirov et al., 2013, Jaffe et al., 2020, Nakahara et al., 2019, Tavitian et al., 2019) and we
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have discovered here that dysfunction of a protein implicated in schizophrenia leads to
significantly increased glutamatergic synapse function, it is possible that a pathological increase
in glutamatergic synapse function in DG granule neurons may be a common feature of
schizophrenia. Other risk genes implicated in schizophrenia should be investigated in the context
of the dentate gyrus. If this pathological increase is not unique to SAP97/DLG1 but also found
with other schizophrenia risk genes, this could suggest that dentate gyral glutamatergic synaptic
transmission could be a promising new therapeutic target for treating schizophrenia and other
neuropsychiatric disorders.
In such efforts to search for new therapeutic targets to treat synaptic disorders, the other
half of the synapse must also be carefully considered. In Chapter 3, we introduced a novel
optical/electrophysiological method of studying proteins regulating presynaptic glutamatergic
synapse function within intact and endogenous synapses in the mammalian central nervous system.
We utilized our new method to determine the presynaptic role of Syt1 in the Schaffer collateral-
CA1 synapse. We found a significant reduction in Schaffer collateral synapse function following
Syt1 knockdown in CA3 neurons, and we determined that this reduction was only discernible using
optical stimulation, demonstrating the critical need for incorporating optogenetics in our approach.
Our method may now be leveraged to expand our understanding of the presynaptic roles of other
proteins implicated in complex brain disorders. Further, it may also be applied simultaneously with
biolistic transfection of postsynaptic neurons for simultaneous pre- and postsynaptic genetic
control. Such an approach may open doors for a new line of synaptic studies on the roles of specific
isoform combinations of transsynaptic adhesion molecules that have been strongly implicated in
brain disorders.
113
Basic characterizations of proteins implicated in brain disorders like Syt1 have already
successfully led to identification of potential new therapeutic targets. The critical role that Syt1
plays in the brain was recently highlighted by the discovery of the Syt1-associated
neurodevelopmental disorder (Baker et al., 2018, Baker et al., 2015). Following this discovery,
Bradberry et al. characterized the disorder-associated Syt1 mutations and found that the mutations
led to decreased presynaptic calcium sensitivity (Bradberry et al., 2020). From this, they reasoned
that increasing presynaptic calcium influx may mitigate the deficits from these Syt1 mutations.
They employed 4-Aminopyridine (4-AP), a potassium channel antagonist that has already been
clinically approved for use in multiple sclerosis (Leussink et al., 2018), and found that 4-AP largely
mitigated the deficits caused by the Syt1 mutations in a dose-dependent manner. While these
results are still preliminary in clinical relevance, they embody the shared goal of such basic
characterization studies: to identify better therapeutic targets to treat brain disorders. My hope is
that the work described in this dissertation will help enhance our understanding of the synapse,
uncover pathogenic molecular mechanisms, and get us one step closer to identifying more effective
therapeutic targets for brain disorders.

114
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Abstract (if available)

Abstract Dysfunction of glutamatergic neurotransmission underlies the pathophysiology of various complex brain disorders, such as schizophrenia. While the exact etiology of schizophrenia remains to be elucidated, accumulating evidence has implicated the MAGUK protein SAP97 in schizophrenia. However, SAP97’s relevance in glutamatergic neurotransmission has long remained uncertain. In Chapter 2, we employ a targeted approach to uncover SAP97’s synaptic role by first visually identifying the dentate gyrus, a hippocampal region independently implicated in schizophrenia, as where endogenous SAP97 may play a synaptic regulatory role. We find that schizophrenia-related perturbations of SAP97 produce significant augmentation of glutamatergic neurotransmission in the dentate gyrus but not in the CA1 region, where SAP97 was previously studied despite little endogenous expression in this region. Further, disrupting SAP97 function in this region impairs contextual episodic memory in rats, altogether demonstrating that the synapse regulatory mechanism involving SAP97 in this region may contribute to the development of symptoms associated with schizophrenia. Chapter 3 highlights the significance of the other half of the synapse in regulating glutamatergic neurotransmission and maintaining healthy brain function, as mutations in key presynaptic proteins have also been strongly implicated in complex brain disorders. We introduce a novel method designed to expedite our understanding of molecular regulatory pathways that govern presynaptic function, and we utilize this method to determine synaptotagmin 1’s presynaptic role in the Schaffer collateral-CA1 synapse. This new method may now be leveraged to bolster our understanding of the presynaptic roles of other proteins implicated in complex brain disorders. Altogether, this dissertation uncovers the synapse-specific regulatory roles of proteins implicated in complex brain disorders, and my hope is that this work will contribute to the development of new strategies to more effectively treat these disorders.

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Creator Kay, Yuni (author)

Core Title Uncovering synapse-specific roles of proteins implicated in complex brain disorders via novel and targeted approaches

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Degree Conferral Date 2022-05

Publication Date 03/05/2022

Defense Date 02/25/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag dentate gyrus,DLG1,glutamate,glutamatergic synapse,hippocampus,MAGUK,OAI-PMH Harvest,SAP97,schizophrenia,synapse,synaptic transmission

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Advisor Dickman, Dion (committee chair), Arnold, Don (committee member), Herring, Bruce (committee member), Katritch, Vsevolod (committee member)

Creator Email ykay@usc.edu,ykbyul27@gmail.com

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