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The role of Rho-GEF signaling in synapse function and autism-related disorders

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The role of Rho-GEF signaling in synapse function and autism-related disorders

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Content THE ROLE OF RHO-GEF SIGNALING IN SYNAPSE FUNCTION AND AUTISM-
RELATED DISORDERS
By
Chen Tian
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)
December 2021
Copyright 2021 Chen Tian
ii
Acknowledgments
I am extremely grateful for all the support I’ve received throughout the years in graduate
school. I would not accomplish what I’ve done without all the help from my family, my mentor,
lab mates, friends and colleagues, and everyone at USC Neuroscience graduate program.
I have been extremely lucky to have Dr. Bruce E. Herring as my mentor, who guided me
into the field of synapse and shaped the way I think about science. Over the years, with tremendous
enthusiasm and patience, Bruce has helped me improve my scientific knowledge, research ability
as well as my ability to present and communicate my discovery. I would not learn so much without
Bruce keeping a good balance between giving advice and leaving room for innovation. I deeply
appreciate the freedom of pursuing my question of interest with enlightening guidance and great
advice on almost all experiments. Bruce is not only a good science mentor but also a mentor for
life. He’s helped me learn American culture, make life plans, release stress and many more. It is a
wonderful experience growing and learning in Bruce’s lab.
I would like to gratefully thank other members of my Ph.D. guidance and dissertation
committee. I have learned a lot from Dr. Don B. Arnold, Dr. Dion K. Dickman, Dr. Samuel
Andrew Hires and Dr. Marcelo Pablo Coba for inspiring advice and kindly support in scientific
research and career advice through my PhD. I also owe a huge thanks to our collaborators who
helped me through all of my projects. I thank Dr. Vsevolod Katritch and Anastassia Sadybekov
for their valuable insight of protein structure and computational modeling. I also deeply appreciate
Dr. Katherine Roche and Jeremiah D. Paskus for their informative protein interaction analysis and
great help on our collaboration.
I’d like to give special thanks to the members of the Herring lab. I have obtained a lot of
good advice, confidence as well as strength with all the love and help from Yuni Kay, Sadhan Rao,
iii
Anna Pushkin and Manual Cerpas Lua. I cannot say enough ‘thank you’ to all of the lab members
who have made grad school so much more enjoyable and more fun. I want to express special thanks
to Yuni Kay, with whom I worked closely, for being my friend, editing scripts with me, teaching
me biochemistry skills and supporting me unconditionally. I would also like to thank Anna Pushkin
for always being extremely supportive, sharing valuable ideas for science and presentation, being
inspirational and helpful throughout all times. I would like to thank Sadhna Rao for creative and
valuable discussions in research, sharing experience and troubleshooting in research as well as life.
I sincerely want to give special thanks to Manual Cerpas Lua for all the skillful and hard work. I
won’t be able to make so much progress without all the help from Manny. It is a great pleasure to
work with such an intelligent and nice group of people.
I’d also like to thank all the support from friends and colleagues from the Neuroscience
Graduate Program, especially the Neurobiology section. I’ve obtained inspiring research opinions
from seminars and neurobiology section retreats. I’ve also earned technical and mental support
from colleges and our special staff Jessica Alaron, Morgan Nagatani, Deanna Solorzano, Dawn
Burke and Melissa Salido.
Very importantly I’d like to deeply thank my dear parents, Feng Wang and Yunliang Tian,
and my boyfriend Athanasios Rompokos for their unconditional support and love. I am deeply
thankful for all the encouragement for hard work and for helping me figure out and pursuing my
dream. The faith from family helped me build my faith in myself, my PhD career and in my future.
iv
Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables ................................................................................................................................... vi
List of Figures ................................................................................................................................ vii
Abbreviations ................................................................................................................................. ix
Abstract ............................................................................................................................................x
Chapter 1: Introduction ....................................................................................................................1
1.1 Brief introduction of autism spectrum disorder .............................................................1
1.2 Genetics of autism spectrum disorders ..........................................................................2
1.3 Introduction of synapse ..................................................................................................4
1.4 Synapse alteration in autism ..........................................................................................8
1.5 Disrupted synapse function in Autism animal model ....................................................9
1.6 Convergence of ASD risk genes and ASD signaling pathway ....................................15
Chapter 2: An Autism Spectrum Disorder-related De Novo Mutation Hotspot Discovered In The
GEF1 Domain of Trio ...................................................................................................16
2.1 Abstract ........................................................................................................................16
2.2 Introduction ..................................................................................................................16
2.3 Materials and methods .................................................................................................18
2.4 Results ..........................................................................................................................24
ASD-related mutations in Trio...............................................................................24
ASD mutations in Trio are predicted to alter Rac1 activation ...............................29
Trio-9 I1329HLAL* expression inhibits synaptic function...................................32
Trio-9 K1431M expression inhibits synaptic function ..........................................36
Trio-9 P1461T expression inhibits synaptic function ............................................41
Trio-9 D1368V results in Trio hyperfunction ........................................................44
2.5 Discussion ....................................................................................................................48
v
Chapter 3: An Intellectual Disability- related Missense Mutation in Rac1
Prevents LTP Induction ...............................................................................................54
3.1 Abstract ........................................................................................................................54
3.2 Introduction ..................................................................................................................54
3.3 Materials and methods .................................................................................................56
3.4 Results ..........................................................................................................................59
A severe ID-related de novo mutation is predicted to prevent Rac1 activation ....59
Rac1 C18Y inhibits synaptic function ...................................................................61
Rac1 C18Y prevents GTP-mediated activation of Rac1 .......................................67
Rac1 C18Y prevents LTP induction ......................................................................74
3.5 Discussion ....................................................................................................................74
Chapter 4: Autism Spectrum Disorder/Intellectual Disability-associated Mutations in Trio Disrupt
Neuroligin 1-m ediated Synaptogenesis .......................................................................78
4.1 Abstract ........................................................................................................................78
4.2 Introduction ..................................................................................................................79
4.3 Materials and methods .................................................................................................80
4.4 Results ..........................................................................................................................83
The ASD/ID-related mutation Trio N1080I disrupts Trio protein function and
blocks synaptogenesis mediated by the ASD/ID-related mutation Trio D1368V.
................................................................................................................................83
Trio N1080I inhibits Trio’s interaction with NLGN1 and blocks NLGN1-mediated
synaptogenesis. ......................................................................................................88
Trio N1080I prevents NLGN1 from increasing NMDAR- but not AMPAR-
mediated synaptic transmission. ............................................................................92
NLGN signaling is required for Trio D1368V-mediated synaptogenesis. ...........98
4.5 Discussion ..................................................................................................................102
Chapter 5: Conclusion and discussion .........................................................................................106
References ....................................................................................................................................110
vi
List of tables
Table 2.1: Predictions of free energy change in Trio GEF1/DH1
domain stability and binding to Rac1 ...........................................................................31

vii
List of Figures
Figure 2.1: ASD-related de novo mutations in Trio ......................................................................25
Figure 2.2: Trio mutations found in control and ASD-related genomes .......................................26
Figure 2.3: Missense and Synonymous mutations found in Trio in the 1000 genomes database .28
Figure 2.4: Predicted effects of ASD-related DH1 mutations on Rac1 activation ........................30
Figure 2.5: Trio-9 p.I1329HLAL* expression reduces the strength of glutamatergic synapses ...33
Figure 2.6: Trio-9 p.K1431M expression reduces the strength of glutamatergic synapses ...........37
Figure 2.7: Trio-9 p.S1575N does not alter Trio-9 mediated potentiation of synaptic AMPAR
expression ......................................................................................................................................39
Figure 2.8: Trio-9 p.K1461T expression reduces the strength of glutamatergic synapses ............42
Figure 2.9: Trio-9 p.D1368V produces a hyperfunctional synaptogenic form of Trio .................45
Figure 3.1: A de novo missense mutation in the P-loop region of Rac1 in an individual with severe
intellectual disability (ID) is predicted to prevent Rac1 activation ...............................................60
Figure 3.2: Rac1 C18Y weakens glutamatergic synaptic transmission .........................................62
Figure 3.3: The ID-related mutation C18Y prevents synaptic potentiation by a constitutively active
form of Rac1, Rac1b ......................................................................................................................68
Figure 3.4: Rac1 C18Y expression prevents long-term potentiation (LTP) induction ..................72
Figure 4.1 The ASD/ID-related mutation Trio-9 N1080I disrupts Trio protein function and blocks
synaptogenesis mediated by the ASD/ID-related mutation Trio-9 D1368V .................................84
Figure 4.2 Trio-9 N1080I inhibits Trio’s interaction with NLGN1 and blocks NLGN1 mediated
synaptogenesis ...............................................................................................................................89
Figure 4.3 Trio-9 N1080I prevents NLGN1 from increasing NMDAR but not AMPAR-mediated
synaptic transmission .....................................................................................................................93
viii
Figure 4.4 NLGN signaling is required for Trio-9 D1368V-mediated synaptogenesis ................99

ix
Abbreviations
ASD, autism spectrum disorder
aCSF, artificial cerebrospinal fluid
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
CaMKII, Ca2+/calmodulin-dependent protein kinase
CNV, copy number variation
Co-IP, co-immunoprecipitation
DH, Dbl homology
Dbl, diffuse B cell lymphoma
eEPSC, evoked excitatory postsynaptic currents
FRET, fluorescence resonance energy transfer
FLIM, fluorescent life time imaging
GST, glutathione S-transferase
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
ID, intellectual disability
LTP, long-term potentiation
LC-MS/MS, label-free liquid chromatography with tandem mass spectrometry
miR, microRNA
NMDA, N-Methyl-d-aspartic acid
PH, pleckstrin homology
PFA, paraformaldehyde
PSD, postsynaptic density
RhoGEF, Rho guanine nucleotide exchange factor
Rac1, Ras-related C3 botulinum toxin substrate 1
SH3, src Homology-3
x
Abstract
Approximately 1 in 54 children in the US is affected by autism spectrum disorder (ASD).
There is a strong genetic basis for ASD but the risk architecture is very heterogeneous. Genetic
studies revealed that accumulative ASD risk genes encode proteins essential in synapse function.
And a growing body of evidence shows alteration in synapse function in human patients as well
as animal models. Glutamatergic synapses, the most common excitatory synapses, are formed on
mushroom-like protrusions, dendritic spines. These dendritic spines are filled up and supported by
a dense skeletal meshwork of actin filaments. The small GTPase Rac1, a key regulator of actin
polymerization, has been implicated as a convergence point of many ASD risk genes.
Here we have identified a large cluster of ASD mutations in the Rac1 activating domain of
Trio. The large number of ASD-related mutations we find in the small domain results in a very
high genome-wide statistical confidence that these mutations contribute to ASD. We revealed that
the majority of these mutations eliminated Rac1 activation and weakened synapse transmission.
One mutation D1368V distinctively enhanced glutamatergic synaptogenesis. Downstream of Trio,
we characterized a severe ID-related mutation C18Y in Rac1. The mutation C18Y disrupts GTP-
mediated Rac1 activation and prevents the induction of long term potentiation (LTP), the cellular
mechanism involved in learning and memory. Upstream of Trio, we found that ASD risk gene
Neuroligin-1 regulates synapse function through its interaction with Trio and its paralog Kalirin.
We found that ASD-related mutation N1080I in Trio disrupted interaction between Trio and
Neuroligin and prevented Neuroligin mediated synaptogenesis. The synaptogenic hyper functional
ASD-related mutation Trio D1368V rely on Neuroligin1 signaling to pathologically enhance
synaptogenesis. Together our findings point to the “Neuroligin-Trio-Rac1” pathway as a possible
key pathway that contribute to the development of ASD-related disorders.
1
Introduction
1.1 Brief introduction of Autism spectrum disorder
Autism spectrum disorders (ASDs) are a complex set of developmental disorders
characterized by impairment in social interaction and communication, often accompanied by
restricted and repetitive behaviors. CDC reported in 2020 that approximately 1 in 54 children in
the U.S. is diagnosed with ASD, with boys being four times more likely to be diagnosed compare
to girls.
The concept of autism originated in 1911 when the German psychiatrist Eugen Bleuler
coined the term autism to describe ‘hallucinations and unconscious fantasy life in infants’, a
symptom of the most severe cases of schizophrenia
1
. Later on in 1943, Leo Kanner first
systematically reported the conditions of autistic children in his classic paper autistic disturbance
of affect contact
2,3
. His deliberate clinical description illustrated critical features such as ‘insistence
on sameness’ and ‘extreme aloneness’ which continue to be included in diagnostic criteria today
4
.
Kanner’s cases were however classified as ‘childhood schizophrenia’ during 1940s and 1950s
when schizophrenia and autism were largely interchangeable. Beginning in the 1960s British
psychologist started to challenge Bleuler’s assumptions about infantile thought and reformulated
description of autism. In exact opposite of ‘excess hallucination and fantasy’, in 1970s the word
‘autism’ referred to ‘complete lack of an unconscious symbolic life’
1
. The change was later
reflected in DSM-III (3
rd
edition of the Diagnostic and Statistical Manual of Mental Disorders), in
which ‘childhood schizophrenia’ was taken out and new category of ‘pervasive developmental
disorders’ including ‘ infantile autism’ was introduced
3
. Criteria were broaden in DSM-IV and
more genetic syndromes including Rett syndrome was included. The current edition DSM-5
2
included more than 20 genetic syndromes under the umbrella category ‘autism spectrum disorder’
(ASD) and provided standard criteria of diagnosing ASD.
Despite the increased attention and diagnosis in ASDs, the pathogenesis of ASDs has
remained a mystery. ASDs are not distinct disorders but instead a complex spectrum of social and
communication impairment and behavioral repetition
5-7
. Many believed that ASDs are caused by
combination of environmental and genetic factors. Direct and indirect evidence has shown the
contribution of environmental factors including prenatal and perinatal factors, socioeconomic
factors as well as drug and toxic exposure
8,9
. Twin studies and family studies, on the other hand,
strongly suggested that autism is predominantly a genetic disorder
6,10,11
. The British twin study in
1995 illustrated a 60%-90% concordance rate in monozygotic(MZ) twins in contrast to 0%-10%
rate in dizygotic(DZ) twins
11
. It is now widely accepted that Autism has a strong genetic basis and
genetic research has been playing a leading role in understanding etiology of autism.
1.2 Genetics of autism spectrum disorders
Advance in diagnosis and assessment as well as technology facilitated large-scale autism
genetic studies. Early genetic studies rules out the possibility that single gene would carry large
effect on autism. Instead, genetic studies illustrated the heterologous risk architecture: ASDs could
be caused by multiple different variation in DNA sequence or structure.
Initially, it had been taken for granted that as the ASDs are a group of common disorders,
there must be common genetic factors contributing to the ASDs. This is referred to as common
disease-common variant hypothesis. Whole-genome linkage studies and association studies were
therefore applied to detect common risk alleles. Successful research has point to genes or loci of
interest such as NLGN4
12
, regional containing MECP2
13,14
and CNTNAP2
15,16
. Despite the
notable success, very few linkage were reported in the literature. Moreover, these common alleles
3
are reported to carry very small risk
17
. There is even doubt in ‘whether any of the common variants
identified thus far, each only subtly modulating risk, have added anything other than confusion to
an otherwise productive field’
10
.
While genetic variation effect phenotype of each individual, natural selection is one of the
major factors that determine the frequency of the variants. If a genetic variant causes death or
reduce the capacity of being reproductive, it is less likely to reach a high frequency in the
population. ASDs are a spectrum of developmental disorders, begin in the first few years of life
and affect the ability in communication and interaction. This early onset and reduction in social
communication suggest a reduction in reproductive fitness, therefore a less likelihood for the
genetic variant to propagate in the population. Together these indicates a rare variant-common
disease model for ASDs
18
and more challenge in identifying genetic variants
19
.
Indeed, mounting evidence has shown successful discovery and the importance of rare
variants in ASDs. Cytogenetic studies was one of the first to show contribution of rare
chromosomal abnormalities to ASDs phenotype
20,21
. Despite the low genomic resolution with the
early techniques, these cytogenetic studies provided region of interest for further candidate
resequencing. Later, with the escalation in genomic resolution offered by new array-based
approaches, significant association were found between de novo copy number variations (CNV)
and ASDs
10,22-24
. De novo CNVs were found in 10% from simplex families (autism in children
with no family history), 2% from multiplex families (patients with an affected first-degree family)
and 1% individuals from control families
10,22
. Most of the detected CNVs were smaller than
microscopic resolution, some of which are even mutations of single genes
22
. However, even with
the increase in resolution, in most cases de novo copy number events span many genes. The
accuracy was boosted by the astonishing development of high-throughput DNA sequencing in the
4
past decade, which made it possible to search for de novo mutations in single genes. Large number
of sequencing studies has been carried out since then, yielded a promising number of new
candidate ASD-risk genes
25-29
.
The next-generation sequencing and the revolutionized discovery of new ASD risk genes
offers a potential new insight into neurobiology and etiology of ASD. And there comes the great
challenge in understanding the clinical contribution and neurobiological consequences of the large
number of genetic variances. Such work is very likely to be essential in decoding the genetic and
phenotypic heterogeneity of ASD. Of the identified ASD risk genes, two major subgroups has
been identified: genes involved in transcription regulation and genes encoding proteins directly
regulate synapse transmission
29
. Although transcription factors could lead to broad effect therefore
hard to characterize, evidence has shown activity of them regulated by neuronal activity and their
effects on neuronal function and synapse maturation
30-33
. Therefore it is essential to understand the
regulation of synapse function and the effect of these ASD risk genes on synapse transmission in
order to further make biological sense of the ASD related genetic variants.
1.3 Introduction of synapse
The rapid and delicately regulated communication between neurons supports the signal
propagation in the brain. Synapse is a fundamental component where one neuron interface and
communicate with another. Most of the connection in the central nervous system rely on the
process during which neurotransmitters release from the presynaptic terminal, travel across the
synaptic cleft and activate ionotropic receptors on the postsynaptic membrane. The process is
called synapse transmission.
Synapse can be excitatory or inhibitory, depending on the outcome of the activation of
ionotropic receptors. In excitatory synapse activation of receptors bring the postsynaptic potential
5
closer to the firing threshold. Vice versa in inhibitory synapses activation of receptors drive
potential away from firing threshold. Most excitatory synapses in the brain are glutamatergic
synapses where neurotransmitter glutamates activate postsynaptic glutamate receptors. Glutamate
receptors contains two broad categories: ionotropic and metabotropic glutamate receptors
(mGluRs). The ionotropic glutamate receptors such as AMPAR and NMDAR are ligand gated
channels where glutamate binding directly opens the channel and quickly relays information.
mGluRs on the other hand indirectly open channel through signaling cascade via G protein-
coupled receptors and associate with prolonged stimulus. Inhibitory synapses are largely mediated
by neurotransmitter -aminobutyric acid (GABA) or glycine. GABA is the major inhibitory
transmitter in the brain and spinal cord, which acts on ionotropic receptor GABAA and
metabotropic receptor GABAB. Glycine alternatively is the major inhibitory transmitter in the
spinal cord and less common in the central nervous system.
Excitatory and inhibitory synapses not only differ in neurotransmitters and receptors but in
structure as well. Inhibitory synapses are generally formed on the shaft of dendritic branches while
excitatory synapses primarily formed on “mushroom like” membrane protrusions from the
dendrites called dendritic spines. The dendrites then integrate synaptic signals in a nonlinear
manner
34
. While the differentiation between excitation and inhibition describes whether synapses
cause a positive or negative effect, the strength of synapses define the weight of each stimulus.
Importantly the strength of synapse could be modulated overtime due to distinct activity
experience, which allows modification of neural circuit function. This ability of synapse to
strengthen or weaken is called synaptic plasticity.
There are many forms and mechanisms of synaptic plasticity. NMDAR dependent long
term potentiation (LTP) and long term depression (LTD) in glutamatergic synapses are by far the
6
most extensively studied and therefore the prototypic forms of synaptic plasticity. LTP is a
persistent strengthening of synapses following a high-frequency stimulation and as an opposing
process LTD is an low-frequency activity dependent reduction in the synapse strength. The
functional alteration in synapse strength is often accompanied by change in synapse morphology.
Using novel technique two-photon glutamate uncaging, enlargement of stimulated spines has been
visualized following repetitive quantum-like uncaging of glutamate
35
.
Synaptic plasticity LTP and LTD are mediated and regulated by a variety of synaptic
proteins. For example the alteration in synaptic strength is primarily due to postsynaptic
modification in ionotropic receptors AMAPRs. Ionotropic receptors NMDARs are also essential,
as activation of NMDARs during postsynaptic depolarization is required for LTP and LTD.
AMPARs and NMDARs are anchored and organized at the synapse in the postsynaptic density
(PSD) by scaffolding proteins like MAGUKs
36
. Removing MAGUKs proteins from neurons leads
to significant reduction in synaptic transmission and multiple evidence has pointed to the
importance of MAGUKs in LTP and LTD
37-39
. In addition to docking receptors, MAGUKs
proteins also interact with other critical components, such as cell adhesion molecules (CAMs)
40
.
CAMs are transmembrane proteins that align presynaptic and postsynaptic neurons. After
forming the physical connection between pre- and postsynaptic, the CAMs mediate signaling
across the synapse and plays an important role in the maturation of the synapses
41-43
as well as
regulation of synaptic strength
44,45
. Despite the lack of knowledge in how exactly CAMs provide
information required for synapse formation, studies has shed light on CAMs mediated regulation
of essential proteins at PSD. The direct interaction between the best studied CAM Neuroligin and
MAGUK protein PSD95 for instance suggests a role of CAMs in recruitment of MAGUK proteins
to the PSD
40
. Such process is followed by recruitment of other functional components to the PSD.
7
In addition to distribution of functional elements, a recent discovery of direct interaction between
Neuroligin and Kalirin suggests CAMs’ role in regulating synapse structure
46
. Almost all synapses
form initially on newly extended dendritic filopodia and increase in synaptic strength is closely
correlated with physical enlargement of glutamatergic synapses
35,47
. Understanding the effect of
Neuroligin on synapse shape and size may greatly facilitate the comprehension of synapse
development and regulation.
The close correlation of synapse morphology and function also demonstrates the
importance of proteins involved in the rapid structural modification of synapses. Synapses are
filled up and supported by actin filaments. The rapid alteration between F-actin and G-actin and
regulation of actin polymerization are essential for glutamatergic synapses development and
plasticity. With LTP spines size increase with more F-actin; with LTD spines shrink with
depolymerization of F-actin
48,49
. Actin filaments are mainly modulated by the Rho family of small
GTPases such as Rac1, cdc42 and RhoA
49
. These GTPases switch between two states: active state
when bound to GTP and inactive state when bound to GDP. When Rho GTPases are active, Rho
GTPase promote actin polymerization through Rho effectors. The cycling between active and
inactivate state is regulated primarily by two category of regulatory molecules GEFs and GAPs.
GEFs activate GTPase by exchanging GDP for GTP and GAP proteins accelerate GTP hydrolysis
to GDP.
Growing number of these actin modulators have been reported to be involved in synapse
development and plasticity. Rho GTPases are critical for protrusion formation and stabilization
50
,
and required for both structural LTP and functional LTP
50-53
. Proteins that regulate Rho GTPase
activity are reported to regulate spine formation and synapse plasticity as well. Inhibition of Rho
GEF protein such as PIX, Kalirin-7 and Trio leads to decreased spine number and disruptive
8
synapse plasticity
54-56
. Inhibition of GAP proteins such as Oligophrenin-1 has been reported to
decrease spine length
57
. How actin polymerization contribute to the alteration of synapse strength
is however unclear. There are different models supported by evidences. For example it is possible
that actin polymerization during LTP lead to increase the size and the number of slots for AMPARs
in PSD. It is alternatively possible that actin polymerization during LTP enhance the fusion of
AMPAR-containing vesicles or eliminate AMPAR endocytosis.
Despite the number of questions remained to be investigated, considerable progress has
been made in understand synapse function and plasticity. Future research will certainly bring more
clarification about synapse mechanism, their contribution in brain function as well as the relevance
between disrupted synapse function and psychological disease.
1.4 Synapse alteration in autism
The structure of neuronal circuits that supports cognitive processing in the brain is shaped
and refined during development, driven by spontaneous brain activity as well as environmental
stimuli
58
. The formation and maintenance of the neuronal circuits rely heavily on the precise
control of synapse development and function. Alteration of synapse function could result in
deleterious consequences including memory, sensory and emotion alterations.
Both human and animals studies has illustrated atypical spine morphology and synapse
function in autism. Acquiring detailed information of alteration in spine development in human
psychiatric disease is exceedingly difficult owing to the impracticality to observe spine
morphology in live brain tissue and the limited availability of post-mortem human brain tissue.
However progress has been made. Golgi-impregnated study of cortical pyramidal cells in the
cortex of ASD subjects has shown increase in spine density, predominantly in layer II and within
layer V of the temporal lobe
59
. Consistent with this study Magnetic Resonance Imaging (MRI) has
9
illustrated hyper expansion of cortices as well as cortical gray matter before 2 years old in ASD
subjects
60,61
, suggesting the spine overgrowth during development in Autism.
Alteration in spine morphology has been recorded not only in autism but in a variety of
neuropsychiatric diseases. Abnormality in spine was firstly found in mental retardation when long
thin spines was recorded in cortical neurons in children with retardation back in 1974
62
. In epilepsy
patient dendritic architecture was reported to be simplified in third cortical layer
63
. Similarly
reduction of spine density on pyramidal neurons of prefrontal cortex has been found in
schizophrenia
64
. The atypical dendritic development in neuropsychiatric disease further support
the importance of spine morphology in autism.
Despite the clear correlation between alteration in spine morphology and autism. The
mechanism of synapse dysfunction underlying autism remain largely uncovered. This is mainly
due to the large number of risk genes involved in autism and the complicated genetic architecture.
Despite the confusion in gene network underlying autism, numbers of autism animal models has
been made. These animal models allowed carefully and thoroughly observation of synapse
dysfunction and will reveal key neurological deficits involved in the development of ASD related
behaviors.
1.5 Disrupted synapse function in Autism animal model
ASD animal model with SHANK3 mutant
SHANK3 is a leading ASD risk gene, widely characterized in different models including
stem-cell derived neurons
65,66
and mice models
67-75
. SHANK3 gene codes a synaptic scaffolding
protein in the core of PSD. Similar to other synaptic scaffolding proteins like PSD95, SHANK
proteins are believed to interacts with critical synapse components including CAM neuroligin,
glutamatergic receptors and cytoskeletal proteins and function as an organizer at the PSD.
10
The first evidence linking SHANK3 and autism was described in 2001 during which the
genetic resolution is still low. In the 2001 study 37 patient with 22q13 deletion showed global
developmental delay, absent or delayed speech and minor dysmorphic features
76
. Further
characterization of 22q13 deletion syndrome narrow down the risk region to SHANK3, stating that
SHANK3 (also called ProSAP2) haploinsufficiency is the major causative factor
77-79
. It was only
until recently that studies showed even de novo mutations in SHANK3 are associated with ASDs
including intellectual disability, developmental delay and absent or severe impaired speech
80,81
.
To characterize ASD neuropathology, numerous mouse lines carrying SHANK3 deletions has
been generated
67-75
. Disruption of the SHANK3 gene have resulted in autistic-like behavior
including deficits in social interaction
67,70-75
and repetitive behaviors
67-75
in majority of the mouse
lines. And several mouse lines showed reduced learning ability
69,71,74
and impaired motor
coordination
69-75
. The autistic-like behaviors in the SHANK3 deletion mouse lines allows
investigation into questions like how dysfunction in SHANK3 affect synapse function, and the
most challenging question how is SHANK3 correlate with autistic-like behaviors.
First synapse characterization in SHANK3 mice model was reported in 2010. In the study,
hippocampal slice examination in SHANK3 heterozygous mice showed decrease in synapse
transmission as well as altered synapse plasticity
72
. Hippocampus is essential in learning and
memory and is also one of the best model for synapse investigation. In multiple following
SHANK3 mouse models, similar synapse dysfunction in hippocampus including reduced spine
density, reduction in excitatory transmission and disrupted synapse plasticity were discovered
which further supported the importance of hippocampus synapse function and plasticity in
ASD
71,73,74
.
11
Striatum and striatal-cortical connection has also aroused considerable interest, primarily
due to relevance between basal ganglia and ASD as well as enrichment of SHANK3 in striatum.
The SHANK3 mutant mice showed altered PSD composition such as disruption in mGluR5-
Homer scaffolding
69
and decreased protein level of critical component including glutamatergic
receptors subunits and scaffolding proteins
67,70
. Altered molecular composition implicated change
in synapse structure and function. Indeed significant reduction in spine density as well as reduction
in synapse function was observed in both Shank3B−/− mice
67,70
and homologous SHANK3
mutant mice InsG3680
75
. These alteration in synapse, startlingly, can be reversed by re-expression
of the Shank3 gene in adult mice
70
. In SHANK3
fx/fx
mice (function as SHANK3 knock out),
injection of tamoxifen restores the expression of SHANK3 in the adult mice
70
. Restoration of
SHANK3 level has been shown to restore the protein level in synapse as well as synapse structure
and function. And this restoration of SHANK3 and synapse function accordingly rescued social
interaction and repetitive grooming behavior, suggesting a causal effect of SHANK3 dysfunction
in autistic-like behavior.
ASD risk genes Neuroligins
The male to female ratio in ASD is 4:1, which have aroused considerable interest in sex
chromosome X and Y. X-linked genes NLGN3 and NLGN4 in neuroligin family are reported to
be associated with ASD even before the explosion of next generation sequencing
12,24,66,82,83
.
Neuroligins (NLGNs) are postsynaptic cell adhesion molecules. They bind to presynaptic
neurexins, mediate the signaling between the pre and post synaptic terminal and is essential for
synapse formation and maturation
84,85
.
The relevance between X-linked genes with male to female ratio is owing to the dramatic
damage in Y chromosome over millions of years. But this is not the case for NLGN4. Within the
12
coding region NLGN4Y isoforms share 89-98% sequence identity with homolog NLGN4X
isoforms
86
. NLGN4Y is then very likely capable of compensating for loss of function caused by
NLGN4X mutation. However a recent characterization pointed out that due to one amino acid
difference between NLGN4X and NLGN4Y, NLGN4Y failed to traffic to surface, therefore
cannot support synaptogenesis
87
. NLGN4X carrying ASD mutations showed similar failure in
trafficking and loss of function. This explained why NLGN4Y cannot compensate for the
NLGN4X loss of function and further supported the potential role of X-linked NLGN genes in sex
ratio in ASD.
To characterize the effect of ASD-related mutation on synapse activity, a knock-in mouse
line with ASD mutation NLGN3 R451C was developed
88
. These mice exhibited impaired social
interaction
88,89
as well as stereotypic and repetitive behavior
90,91
, known as core characteristics of
autism. Some unexpected behavior phenotype such as increased ability for special learning
88
were
also observed in these R451C mice.
These changes in cognitive function are accompanied with synapse alteration.
Characterization of the effect of R451C on synapse has yielded different results across the brain.
R451C mice showed increased inhibitory synapse function and no alteration in excitatory synapse
function in somatosensory cortex
88
. Alternatively inhibitory synaptic currents were dampened in
striatal medium spiny neurons in R451C mice
90
. On the other hand excitatory synapse function
was enhanced in hippocampus that R451C mutation strengthened both AMPAR and NMDAR
mediated synaptic transmission
92
. Different changes in different brain area may account for
different behavior phenotypes in the mutant mice. Investigation of these brain circuitries may shed
light on how alteration in different synapses contribute to ASD.
ASD risk gene FMR1
13
FMR1 is another ASD-related gene on the X chromosome. FMR1 encodes the fragile X
mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transportation
and translation at synapses. Mutations in the gene FMR1 could result in the most common
inherited form of intellectual disability Fragile X syndrome (FXS), which is associated with ASD
and a frequent cause of ASD.
To study how FMRP loss of function contribute to mental retardation, FMR1 KO mice was
made back in 1994
93
. The FMR1 knockout mice showed phenotypes generally consistent with
FXS patients including increase in testicular size, disrupted learning and memory as well as altered
sensorimotor integration
93-95
. Examination was then carried out on the FMR1 KO mice. One of the
key finding in FXS patient is that they have more spines and the spines are longer and thinner
96,97
.
This may seem odd as FMRP does not function as synaptic protein and instead is involved in
mRNA transportation and translation. Indeed FMRP is essential in regulating synaptic molecules
therefore critical in synapse signaling. Spine observation in the cortex of the FMR1 KO mice
similarly showed higher spine density while dendritic spines were longer and often thin
94
.
The increase in immature spine number indicated a failure in synapse maturation and
elimination, which often intertwined with impairment in synapse plasticity. LTP and LTD
mechanisms were then investigated in these mice to understand the neurological pathway, among
which mGluR-dependent LTD (mGluR-LTD) is the most popular and best understood theory
98-100
.
Loss of FMRP resulted in an increase in mGluR-LTD in hippocampus
98
. Reduction in mGluR5
expression has even been reported to rescue alteration in plasticity, cortical neuron spine density
as well as some behavior phenotype
101
. More research has been carried out to answer how exactly
FMRP loss of function affect mGluR-LTD process and signaling pathway
102
. Discovery of the
details in the signaling pathway will provide great number of targets for therapeutic intervention.
14
ASD risk gene SynGAP
Advanced sequencing technology has yielded number of mutations, which however are
only recurrent in a small number of genes. A considerable number of ASD de novo missense and
truncation mutations were detected in SynGAP, illustrated an involvement of SynGAP in
autism
103-106
. De novo mutations in SynGAP were also observed in in patients with other ASD-
related disease such as intellectual disability
103,107,108
, epilepsy
103
and schizophrenia
109
, which
further supported the correlation between SYNGAP and neuropsychiatric disorders.
SynGAP is a synaptic Ras GTPase-activating protein (RasGAP). By facilitates hydrolysis
of GTP, SynGAP negatively regulates Ras activity and its downstream Rac activity. Rac is well-
known in regulating the formation of actin branches, the main cytoskeleton supporting spine
structure. Through regulating Ras and Rac, SynGAP plays an critical role in mediating spine
morphology and synapse plasticity.
SynGAP is exceptionally essential in cognitive function that mice model with SynGAP
dysfunction was created even before he discovery of ASD and ID related de novo mutations. The
first behavior phenotype observed in these SynGAP mutant mice was spatial learning deficits
110
.
Later characterization of the SynGAP mutant mice reported similar memory deficits and other
psychiatric disorder related behavior such as social isolation, lack or social memory, hyperactivity
and impaired sensory-motor gating
111-113
. These mice provided opportunity into understanding the
effect of the SynGAP in brain development and the contribution of SynGAP dysfunction in
neuropsychiatric disorders.
SynGAP is essential in regulating spine morphology. Examination showed that SynGAP
dysfunction accelerates the maturation of hippocampal synapse: enlargement in spine size as well
as enhancement in synaptic transmission
113,114
. This facilitation of synapse maturation shorten the
15
time period during which unstable synapse could be modulated according to environment stimulus.
Spines in the SynGAP dysfunction mice were reported to be significantly less motile
113
. Synapse
plasticity, especially LTP is also disrupted in SynGAP dysfunction mice
110,114
. The alteration in
synapse motility and plasticity may account for the memory deficits. Further study will shed light
more information on the effect of SynGAP in neuropsychiatric behaviors.
1.6 Convergence of ASD risk genes and ASD signaling pathway
Human and animal model studies has uncovered several ASD candidate genes as well as
their effect on synapse function and autistic behavior. Moreover increasing number of ASD
candidate genes are reported owing to the explosion of next generation sequencing technology.
This exponential knowledge creation greatly advanced the understanding of autism. At the same
time the knowledge illustrated a complicated genetic architecture. Such complexity highlighted
the need of understanding the critical neural pathways and finding the convergence point of
different causal factors.
Regulator of actin cytoskeleton, the small GTPase Rac1 in particular, has been implicated
as a promising candidate convergence point of a large number of ASD risk factors
115-119
. ASD risk
gene SynGAP regulate synapse function through regulating Rac1 activity. And in SHANK3 ASD
animal model the downstream effector of Rac1 exhibit significant reduction in activity
119
. The
deficits in Rac1 function and actin cytoskeleton in ASD animal models are suggested to be directly
corelated with autism as restoration of actin cytoskeleton rescues the behavior deficits in both
SHANK3 and Fmr1 mice model
118,119
. However the influence of ASD mutations in Rac1 or Rac1
regulator has yet not been explored.

16
Chapter 2 An Autism Spectrum Disorder-Related De Novo Mutation Hotspot
Discovered in the GEF1 Domain of Trio
2.1 Abstract
The Rho guanine nucleotide exchange factor (RhoGEF) Trio promotes actin
polymerization by directly activating the small GTPase Rac1. Recent studies suggest that autism
spectrum disorder (ASD)-related behavioral phenotypes in animal models of ASD can be produced
by dysregulation of Rac1’s control of actin polymerization at glutamatergic synapses. Here, in
humans, we discover a large cluster of ASD-related de novo mutations in Trio’s Rac1 activating
domain, GEF1. Our study reveals that these mutations produce either hypofunctional or
hyperfunctional forms of Trio in rodent neurons in vitro. In accordance with pathological increases
or decreases in glutamatergic neurotransmission observed in animal models of ASD, we find that
these mutations result in either reduced synaptic AMPA receptor expression or enhanced
glutamatergic synaptogenesis. Together, our findings implicate both excessive and reduced Trio
activity and the resulting synaptic dysfunction in ASD-related pathogenesis, and point to the Trio-
Rac1 pathway at glutamatergic synapses as a possible key point of convergence of many ASD-
related genes.
2.2 Introduction
There is a strong genetic basis for ASD, and recent studies now establish an important role
of germline de novo mutation in ASD-risk. De novo mutations identified in large exome
sequencing studies have identified a number of new candidate ASD-risk genes and have ultimately
led to a revised model for causation
120
. Over the past decade, evidence has also grown suggesting
a convergence on altered regulation of glutamatergic synaptic development and function in
ASD
33,121,122
. Disrupted synaptic actin modulation at glutamatergic synapses has been identified
in well-established animal models of ASD, and in some cases has been identified as the underlying
17
cause of ASD-related behavioral phenotypes in these models
116,118,119
. Rac1-mediated synaptic
actin regulation in particular has been implicated in ASD and has been proposed as a likely point
of convergence of many known ASD risk genes
117-119
.
We have recently discovered that the Rho guanine nucleotide exchange factor (RhoGEF)
protein Trio, along with its paralog Kalirin, is required for glutamatergic neurotransmission
56
. Trio
expression in the brain is highest in late prenatal/early postnatal development while Kalirin
expression does not reach its peak until adolescence
123-125
. Multiple isoforms of Trio originating
from a single gene are expressed in the brain
126
. Trio-9, for example, is the predominant isoform
expressed in the cortex and hippocampus. Both Trio and Kalirin reside within the postsynaptic
compartment of glutamatergic synapses referred to as dendritic spines
58,123
. In spines, these two
proteins regulate glutamatergic synapse function through the ability of their GEF1 domains to
promote Rac1-dependent actin polymerization
56,127,128
. Furthermore, we have found that Trio and
Kalirin are targets of CaMKII phosphorylation that are required for the induction of Long-Term
Potentiation (LTP)
56
, the cellular process believed to underlie learning and memory. Thus, these
proteins play fundamental roles in glutamatergic synapse regulation.
Here we identify a large cluster of ASD-related de novo mutations in the Rac1-activation
domain of Trio, GEF1. The degree of mutational clustering that we find in Trio’s GEF1 domain
and the computationally predicted impact of these ASD-related de novo mutations on Trio-Rac1
interactions suggest a strong association of Trio-Rac1 pathway dysregulation in ASD-related
pathologies. Systematic examination of these mutations in Trio-9 reveals both hypomorphic and
hypermorphic mutations that dramatically and bidirectionally affect Trio’s function and Trio’s
influence on glutamatergic neurotransmission in hippocampal CA1 pyramidal neurons. Animal
models of ASD exhibit pathological increases and decreases in glutamatergic
18
neurotransmission
70,129,130
. Our study uncovers ASD-related missense mutations in a single
synaptic Rac1-activating protein that can produce bidirectional alterations of glutamatergic
neurotransmission and thus implicates both reduced and excessive Trio activity and the resulting
synaptic dysfunction in ASD-related disease.
2.3 Materials and Methods
Mutation modeling
The effect of mutations on stability and binding were predicted using the high-resolution
crystal structure of Trio GEF1 complex with Rac1 (PDB code 2NZ8). Calculations were
performed using ICM molecular modeling software (Molsoft LLC). Energy optimization of
mutant protein conformation was performed using biased probability Monte Carlo algorithm. The
free energy change in protein stability ∆∆G stability(1) and protein binding ∆∆G bind (2) was then
calculated as a difference in folding or binding free energies of mutant and wild-type protein:
ΔΔ 𝐺 stability=(Δ 𝐺 mutantfolded−Δ 𝐺 mutantunfolded)−(Δ 𝐺 WTfolded−Δ 𝐺 WTunfolded)ΔΔGstabil
ity=(ΔGfoldedmutant−ΔGunfoldedmutant)−(ΔGfoldedWT−ΔGunfoldedWT) (1)
ΔΔ 𝐺 bind=Δ 𝐺 mutantbind−Δ 𝐺 WTbindΔΔGbind=ΔGbindmutant−ΔGbindWT (2)
Mutations with either ∆∆G bind > 2 or ∆∆G stability > 2 were predicted as disruptive for Trio
activation of Rac1.
Experimental constructs
Human Trio-9 (or Trio-9s in ref
126
) was generated from a Trio-FL cDNA generously
provided by Dr. Betty A. Eipper (University of Connecticut). ASD-related Trio mutations were
made from Trio-9 cDNA using overlap-extension PCR followed by In-fusion cloning (Clontech).
All plasmids were confirmed by DNA sequencing. Trio-9 cDNAs were cloned into a pCAGGs
vector containing IRES-mCherry. A pFUGW vector expressing only GFP was co-expressed with
19
pCAGG-IRES-mCherry constructs to enhance identification of transfected neurons and was used
as a control vector for spine imaging. The Rac1 biosensor construct was within a pTriEx-HisMyc
backbone, has been described previously in ref
131
and was generously provided by Dr. Jaap D. van
Buul (Sanquin).
HEK293 cell transfection
HEK293T cells (ATCC) were cultured in DMEM with 10% FBS in a 37 °C incubator
supplied with 5% CO2. Cells were plated onto 35 mm glass bottom plates coated with Matrigel.
Cells were grown until 50% confluence before being transfected with the Trio-9 expression
constructs together with the Rac1 biosensor using FuGENE HD Transfection Reagent. A Trio-
9/Rac1 biosensor DNA ratio of 1:1 was used. Plated cells were incubated in the transfection
mixture for 16 h, and then replaced with fresh media. HEK293 experiments were conducted ~20 h
after transfection.
Fluorescence lifetime imaging (FLIM)
Fluorescence lifetime images were acquired from HEK293 cells with a Zeiss LSM-780
inverted microscope coupled to a Ti:Sapphire laser system (Coherent Chameleon Ultra II, 80 fs
pulses with repetition rate of 80 MHz) and an ISS A320 FastFLIM
132,133
. A 40X 1.1 NA water
immersion objective (Zeiss Korr C-Apochromat) optimized for 2-P imaging was used. For image
acquisition, the following settings were used: image size of 256 × 256 pixels and scan speed of
12.6 μs/pixel. A short pass dichroic filter (760 nm) was used to separate the fluorescence signal
from the laser light. For the acquisition of FLIM images, fluorescence light was separated into
donor and acceptor fluorescence by a 509 long pass CFP/YFP filter, and then detected by two
hybrid photomultiplier tube detectors (Hamamatsu R10467U-40), one having a CFP 483/32 and
the other a YFP 537/26 band pass filter for the detection of donor and acceptor signal, respectively.
20
FLIM data were acquired from fields of HEK293 cells using VistaVision software from
ISS Inc., and processed by the SimFCS software developed at the Laboratory of Fluorescence
Dynamics (LFD), University of California Irvine. The FRET donor fluorophore was excited at
840 nm. An average power of about 5 mW was used to excite the cells. Calibration of the FLIM
system was performed by measuring the known lifetime of coumarin 6 in 99% ethanol solution,
characterized by a single exponential decay time of 2.55 ns. Typically, the acquisition time to
obtain an average photon count of 100 counts/pixel was about 30 sec. Data analysis was performed
using the phasor approach to biosensor FLIM-FRET detection
134
. Every pixel of the FLIM image
was transformed in one pixel in the phasor plot as previously described and reported in detail
135,136
.
The coordinates g and s in the phasor plot were calculated from the fluorescence-intensity decay
of each pixel of the image by using Fourier transformations. The analysis of the phasor distribution
was performed by cluster identification. We calculated the FRET efficiency trajectory according
to the classical definition of FRET efficiency: 𝐸 =1− 𝜏𝐷 𝐴 / 𝜏𝐷 E=1−τDA/τD (3)
The phasor of the FRET biosensor in the absence of the activator was obtained from an
independent preparation. The phasor corresponding to the quenched donor was calculated
according to the quenching equation (3). The positions of all possible phasors that are quenched
with different efficiencies describe a curved trajectory in the phasor plot. The experimental
position of the phasor of a given pixel along the trajectory determined the amount of quenching
and therefore the FRET efficiency. The contributions of the background and of the donor without
acceptor are evaluated using the rule of the linear combination
134,137
, with the background phasor
and the donor unquenched determined independently. All phasor transformations and the data
analysis of FLIM data were performed using SimFCS software (University of California, Irvine).

21
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. Organotypic slice cultures
were prepared from P6–9 male and female Sprague-Dawley rat pups as described in detail in
Stoppini et al.
138
. Briefly, hippocampi were removed from P6-P9 Sprague-Dawley rats and 400 μm
transverse sections were made using a MX-TS tissue slicer (Siskiyou). Slices were 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
12360-038), horse serum 25%, HBSS (25%) and L-glutamine (1 mM). Media was exchanged
every other day. Sparse biolistic transfections of organotypic slice cultures were performed on
DIV1 as described in detail in Schnell et al., 2002
139
. Briefly, 50 µg total of mixed plasmid DNA
was coated on 1 µm-diameter gold particles in 0.5 mM spermidine, precipitated in with 0.1 mM
CaCl2, and washed four times in pure ethanol. The gold particles were coated onto PVC tubing,
dried using ultra-pure N2 gas, and stored at 4 °C in desiccant. DNA-coated gold particles were
delivered with a Helios Gene Gun (BioRad). Construct expression was confirmed by GFP and
mCherry epifluorescence. Recordings were performed on day in vitro (DIV) 7 slices. All slices
were maintained during recording in room temperature artificial cerebrospinal fluid (aCSF)
containing (in mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose, 4 CaCl2 and 4
MgSO4. aCSF was supplemented with 5 μM 2-chloroadenosine to dampen epileptiform activity,
and GABAA receptors were blocked by picrotoxin (0.1 mM). aCSF was saturated with 95% O2/5%
CO2. The internal whole-cell recording solution contained (in mM): 135 CsMeSO4, 8 NaCl, 10
22
HEPES, 0.3 EGTA, 5 QX-314, 4 Mg-ATP, and 0.3 Na-GTP and pH buffered at 7.3–7.4.
Osmolarity was adjusted to 290–295 mOsm.
Transfected neurons were identified by epifluorescence microscopy. All paired recordings
involved simultaneous whole-cell recordings from one transfected neuron and a neighboring non-
transfected control neuron. Synaptic responses were evoked by stimulating with a monopolar glass
electrode filled with aCSF in stratum radiatum of CA1. The stimulus was adjusted to evoke a
measurable monosynaptic eEPSC in both cells. Synaptic responses were collected with a
Multiclamp 700B amplifier (Axon Instruments, Foster City, CA), filtered at 2 kHz and digitized
at 10 kHz. Peak AMPAR currents were recorded at −70 mV. NMDAR currents were measured
at + 40 mV and were temporally isolated by measuring amplitudes 150 ms following the stimulus,
at which point the AMPAR-eEPSC had completely decayed. In the scatter plots for simultaneous
dual recordings, each open circle represents one paired recording, and the closed circle represents
the average of all paired recordings. In the scatter plot, the x-axis represents the eEPSC recorded
in the control cell, and the y axis represents the eEPSC recorded in the transfected cell. Virtual 1:1
diagonal line is also shown. If the data point falls below the diagonal line, it indicates that the
eEPSC is higher in the control cell. Paired-pulse ratio was determined by delivering two stimuli at
40 ms apart and dividing the peak response of stimulus 2 by the peak response to stimulus 1. Series
resistance was monitored and not compensated, and neurons in which series resistance varied by
25% during a recording session were discarded. No more than one paired recording was performed
on a given slice. Data was collected and analyzed using in-house software in Igor Pro
(Wavemetrics) developed in Dr. Roger Nicoll’s laboratory at UCSF.
Spine density analysis
23
For spine density analysis, control and experimental CA1 pyramidal neurons in
organotypic hippocampal slice cultures made from P6 rat pups were biolistically transfected with
FUGW-GFP and pCAGGS-IRES-mCherry constructs approximately 18–20 h after plating.
Images were acquired at DIV 7 using super-resolution microscopy (Elyra Microscope System,
Zeiss). For use with the available inverted microscope and oil-immersion objective lens, slices
were fixed in 4% PFA/4% sucrose in PBS and washed 3× with PBS. To amplify the GFP signal,
slices were then blocked and permeabalized in 3% BSA in PBS containing 0.1% Triton-X and
stained with primary antibody against GFP (2 μg/ml, Life Technologies A-11122) followed by
washes in PBSTx and staining with Alexa 488-conjugated secondary (4 μg/ml, Life Technologies
A-11034). Slices were further processed with an abbreviated SeeDB-based protocol
140
in an
attempt to reduce spherical aberration. Slices were then mounted in SlowFade Gold (Life
Technologies) for imaging. Z-stacks were made of 30 µm sections of secondary apical dendrites
~30 µm from the soma. Images were acquired with a 100× oil objective (100×/1.46) in SIM mode
using a supplied 42 μm SIM grating and processed and reconstructed using supplied software (Zen,
Zeiss). An experimenter, blinded to the condition of the image, performed image analysis on
individual sections using ImageJ to count spines extending laterally from the dendrite.
Statistics analysis
For the ASD-related mutation statistics in Trio, we used a simple Poisson test, similar to
the one used in Samocha et al.
141
. The expected number of de novo mutations in Trio protein in
4890 individuals with ASD-related disorders was calculated based on gene-specific mutation
probability as determined by Samocha et al. Expected number of de novo mutations in the Trio-
GEF1/DH1 domain was estimated through rescaling of expected number of mutations in Trio with
assumption of equal mutation probability along the gene. A genome-wide significance threshold P-
24
value of < 1 × 10−6 was used. For paired electrophysiological recordings of eEPSC amplitude, a
Wilcoxon Signed Rank Test for paired data was used. Wilcoxon Rank Sum Tests were used to
compare electrophysiological data across independent conditions. Paired pulse facilitation
measurements and spine density data were analyzed using a paired and unpaired Student’s t-test,
respectively. All statistical tests performed were two-sided and with all tests a P-value of < 0.05
was considered statistically significant. All error bars represent standard error measurement.
Sample sizes in the present study are similar to those reported in the literature.
2.4 Results
ASD-related mutations in Trio
Recent whole-exome sequencing studies have proved fruitful in uncovering risk-conferring
variations, primarily by enumerating de novo variations, which are sufficiently rare that multiple
mutations in a gene suggest a link to ASD. We queried several large databases of de novo
mutations found specifically in individuals with ASD or ASD-related disorders (i.e. intellectual
disability and neurodevelopmental disorders), looking for ASD-related mutations in either Trio or
Kalirin
27-29,120,142,143
. Together these studies included 4890 individuals with ASD-related disease.
We found no ASD-related mutations in Kalirin but found a surprisingly large number of highly
clustered ASD-related mutations in Trio’s GEF1 domain, specifically within the DH1 subdomain
(GEF1/DH1) (Figure 2.1). Trio’s GEF1/DH1 subdomain binds directly to Rac1 and is essential
for Trio’s ability to promote actin polymerization
144,145
. The GEF1/DH1 subdomain of only 175
amino acids harbors six de novo missense mutations (Figure 2.1A), as well as a single nucleotide
deletion resulting in the formation of a stop codon inside Trio’s GEF1/DH1 subdomain
(Figure 2.1B). In addition, another individual was identified where a 16 exon deletion of
the TRIO gene results in the removal of the entire GEF1 domain (Figure 2.1C). We also found two
25

Figure 2.1
ASD-related de novo mutations in Trio.
A, Missense, B, nonsense and C, copy number variation mutations in Trio found in individuals
with ASD-related disorders. The different protein domains are indicated, starting with the N-
terminus: Sec14 domain, Spectrin repeats, GEF1 domain (composed of a Dbl homology domain
(DH1) and a Pleckstrin homology domain (PH1)), Src homology 3 domain (SH3), and the GEF2
domain (composed of a Dbl homology domain (DH2) and a Pleckstrin homology domain (PH2)).
For each mutation, the individual’s diagnosis is given along with information about the alteration
of Trio’s amino acid sequence. Position of amino-acid mutations from NP_009049.2
26

Figure 2.2
Trio mutations found in control and ASD-related genomes.
A, Trio-9 mutations found in control genomes (from De Rubeis et al.
29
). B, The graph shows the
number of ASD-related (red bars) and control (black bars) mutations found in each domain of
Trio-9 in individuals with ASD-related disorders and control individuals without ASD-related
disorders, respectively. ASD-related Trio mutation enrichment in each domain of Trio was
calculated by subtracting the number of control mutations from the number of ASD-related de
novo mutations observed in each domain. Transparent triangles illustrate the presence (red),
absence (black) and magnitude of ASD-related mutation enrichment in each domain.
27
missense mutations in the removal of the entire GEF1 domain (Figure 2.1C). We also found two
missense mutations in Trio’s spectrin repeat region and one missense mutation in the PH
subdomain of Trio’s GEF2 domain (Figure 2.1A). None of these mutations were observed in the
genomic sequences of family member controls
27-29,120,142,143
.
According to the recent Exome Aggregation Consortium (ExAC) analysis, based on 60,706
fully sequenced human genomes, TRIO is one of the top 60 most constrained human genes,
suggesting a high functional importance. Trio has an exceptionally low rate of both missense
mutations (z = 6.29) and loss of function (LoF) nonsense mutations (pLi = 1.), while having an
average rate of synonymous mutations (z = 0.19)
146
. Even more striking is the constraint in the
GEF1/DH1 domain. Remarkably, we observed no missense or loss of function (LoF) mutations in
the GEF1/DH1 domain in 9,937 control genomes
29
(Figure 2.2A), thus indicating a strong
enrichment of ASD-related mutations in this region (Figure 2.2B). We also found no missense
mutations in the GEF1/DH1 domain in the 1000 Genomes database
147
(Figure 2.3A). Importantly,
synonymous mutations listed in the 1000 Genomes database were present in this region
(Figure 2.3B). Taken together, these data point to a strong enrichment of ASD-related de novo
mutations in the GEF1/DH1 region of Trio.
To assess significance of the Trio protein and Trio-GEF1/DH1 domain mutations in ASD-
related disorders, we compared the expected number of de novo mutations to that observed. We
used the mutational model developed by Samocha et al.
141
. The results of Samocha et al. were
based on 1078 ASD cases and 151 cases of intellectual disability, and this study was able to
identify SCN2A and SYNGAP as significant in ASD-related disorders. In the present study, our
much larger case number (4890 for ASD-related disorders) and the large number of missense
28

Figure 2.3
Missense and synonymous mutations found in Trio in the 1000 genomes database
A, Illustration of the positions of all missense Trio mutations found in individuals in the 1000
Genomes database1. Trio- 9 S1575N (in blue) was tested for an effect on Trio-9 function
B, Illustration of the positions of all synonymous Trio mutations found in individuals in the 1000
Genomes database.

29
mutations identified in TRIO (11 ASD cases, as compared to 1.07 cases expected for the same
number of individuals in the general population) dramatically improved the statistics, leading to a
highly significant whole genome association of TRIO with ASD-like syndromes (P-value
of < 1.96 × 10
−8
). We found an even stronger association with ASD-related syndromes for the Rac1
interacting GEF1/DH1 subdomain of TRIO (7 cases compared to 0.06 expected, P-
value < 5.5 × 10
−13
).
ASD mutations in Trio are predicted to alter Rac1 activation
The clustered nature of Trio’s de novo mutations affecting the GEF1/DH1 subdomain
suggests a connection to ASD. While the nonsense and copy number mutants (Figure 2.1B-C)
would apparently result in loss of Trio interactions with Rac1, the effects of the missense mutations
(Figure 2.1A) remain to be identified. We used structure-based modeling to assess the possibility
that these mutations disrupt the ability of Trio’s GEF1/DH1 domain to activate Rac1. The structure
of Trio’s GEF1 domain in complex with Rac1 has been solved
144
(Figure 2.4), which allows
accurate conformational modeling and evaluation of mutation effects on stability and Rac1 binding
(Figure 2.4 and Table 2.1). Of the six missense mutations identified related to Trio’s GEF1/DH1
domain, five were predicted to disrupt GEF1-mediated Rac1 activation (Table 2.1). Two of these
mutations (R1312W and R1428Q) were predicted to destabilize the 3D structure of Trio’s DH1
subdomain due to internal clashes and disruption of the internal hydrogen bonding network. The
other three mutations (K1431M, P1461T and P1461L) were predicted to directly interfere with the
ability of Trio’s DH1 subdomain to interact with Rac1 due to disruption of the intermolecular
hydrogen bonding network (K1431M) or changing the backbone conformation of the protein at
the interaction interface (P1461T and P1461L) (Figure 2.4 and Table 2.1). All five of these
30

Figure 2.4
Predicted effects of ASD-related DH1 mutations on Rac1 activation.
The GEF1 domain in the context of the entire protein is shown above with the GEF1 domain
identified by a dashed red square. An overall view of the Trio-GEF1 and Rac1 complex structure
is shown beneath, with the two interacting proteins shown as blue and orange cartoons,
respectively (Protein Data Bank code 2NZ8). Amino-acid residues mutated in ASD-related disease
are shown as spheres with carbon atoms colored magenta. Zoomed in inserts represent interactions
between amino-acid residues in wild-type and mutant protein. Wild-type residues are labeled and
shown in stick representation, with carbon atoms colored in magenta. Mutated amino acids are
shown with carbon atoms colored green or yellow. Hydrogen bonds/salt bridges are shown
as dashed lines. Water molecules involved in GEF1-Rac1 interactions are indicated by transparent
red spheres.
31

Table 2.1
Predictions of free energy change in Trio GEF1/DH1 domain stability and binding to Rac1
Predictions of free energy change in protein domain stability (∆∆G stability) and binding of Trio-
GEF1 and Rac1 (∆∆Gbind) for mutant proteins and expected effects of mutations on the ability of
Trio to activate Rac1. Starred ∆∆ values indicate predicted inhibition of Rac1 activation. Position
of amino-acid mutations from NP_009049.2 and position of corresponding nucleotide mutations
from Human (GRCh37) or NM_007118.2.

32
mutations were found to be within 6 Å from the Rac1-interacting interface of the GEF1/DH1
subdomain. One notable exception among the GEF1/DH1 mutations, however, was D1368V,
which is located away from the Rac1 interface, does not make any intramolecular interactions, and
was predicted to not impact Rac1 activation. Here we find that this mutation has neurobiological
consequences distinct from the other ASD-related GEF1/DH1 mutations characterized below.
Modeling data along with available patient information were used to select specific ASD-related
missense mutations for further study.
Trio-9 I1329HLAL* expression inhibits synaptic function
The first mutation we examined was Trio-9 I1329HLAL* (Figure 2.5A). This nonsense
mutation, found in an individual diagnosed with ASD, results in truncation of Trio inside its
GEF1/DH1 subdomain and eliminates the Rac1 interface of this region. To examine the impact
this mutation has on the ability of Trio-9 to activate Rac1 we used a recently developed
fluorescence resonance energy transfer (FRET)-based biosensor that measures the ability of
RhoGEFs to activate Rac1
148
. Initially we transfected HEK293 cells with this Rac1 biosensor and
wild-type Trio-9. Using fluorescence lifetime imaging (FLIM) to detect FRET, we found that
HEK293 cells co-expressing wild-type Trio-9 and this biosensor exhibited a much higher FRET
signal relative to cells expressing the biosensor alone (Figure 2.5B). In the representative cell color
map images, colors range from blue (low Rac1 biosensor activation) to red (high Rac1 biosensor
activation). We observed a larger population of quenched donor molecules in the pixels located
toward the cell perimeters compared to the inner region of the cells, indicating a higher level of
Rac1 activity at these locations. In contrast to wild-type Trio-9, the FRET signal observed in
HEK293 cells co-expressing the biosensor and Trio-9 I1329HLAL* was very low (Figure 2.5B).
The near complete lack of Rac1 biosensor activation observed in Trio-9 I1329HLAL* expressing
33

Figure 2.5
Trio-9 p.I1329HLAL* expression reduces the strength of glutamatergic synapses.
A, Illustrations of Trio-9 and Trio-9 I1329HLAL*. B, Trio-9 I1329HLAL* inhibits Trio-9’s
ability to activate Rac1. Representative FLIM color maps of HEK293 cells expressing the Rac1
34
biosensor, the biosensor and Trio-9 I1329HLAL* or the biosensor and Trio-9 are shown above.
A cropped phasor plot for each condition is shown below. Dashed ovals identify the lifetime
distribution for each condition. C, Electrophysiological recording setup. D-I, Scatterplots show
eEPSC amplitudes for single pairs of control and transfected neurons (open circles). Filled
circles show mean ± SEM. (Insets) Current traces from control (black) and transfected (green)
neurons (Scale bars: 20 ms for AMPA, 50 ms for NMDA, 20 pA). D, Trio-9 expression increased
AMPAR-eEPSC amplitude (n = 7 pairs, *P < 0.05, Wilcoxon Signed Rank Test). E, Trio-9
expression did not affect NMDAR-eEPSC amplitude (n = 6 pairs, p > 0.05, Wilcoxon Signed Rank
Test). F, Trio shRNA expression reduced AMPAR-eEPSC amplitude (n = 6 pairs, *P < 0.05,
Wilcoxon Signed Rank Test). G, Trio shRNA expression did not affect NMDAR-eEPSC
amplitude (n = 5 pairs, P > 0.05, Wilcoxon Signed Rank Test). H, Trio-9 I1329HLAL* expression
reduced AMPAR-eEPSC amplitude (n = 7 pairs, *P < 0.05, Wilcoxon Signed Rank Test). i Trio-9
I1329HLAL* expression did not affect NMDAR-eEPSC amplitude (n = 7 pairs, P > 0.05,
Wilcoxon Signed Rank Test). J-K, Average eEPSC amplitudes (±SEM) of neurons expressing
Trio-9, Trio shRNA and Trio-9 I1329HLAL* normalized to their respective average control
eEPSC amplitudes. A Wilcoxon Rank Sum Test was used to compare across independent
conditions, (i.e., Trio-9 and Trio-9 I1329HLAL* in J, *P < 0.05). L, Mean ± SEM paired-pulse
facilitation (PPF) ratios for Trio-9 I1329HLAL* expressing and paired control neurons (n = 6
pairs, P > 0.05, Student’s t-test). Peak 1-scaled current traces from control (black) and transfected
(green) neurons (Scale bar: 20 ms). n.s., not significant. M, CV analysis of AMPAR-eEPSCs from
pairs of control/Trio-9 I1329HLAL* neurons. CV
−2
is graphed against ratio of mean amplitude
within each pair (open circles). Filled circle shows mean ± SEM. (n = 7 pairs)

35
cells looked very similar to those cells that were transfected with the biosensor alone (Figure 2.5B).
Thus, Trio-9 I1329HLAL* dramatically reduces Trio-9-mediated Rac1 activation.
We were then interested in the impact Trio-9 I1329HLAL* expression has on
glutamatergic synapse function. First, we used biolistic transfection to express wild-type Trio-9 in
CA1 pyramidal neurons of organotypic rat hippocampal slice cultures. 6 days after transfection
recordings of AMPA receptor and NMDA receptor-evoked excitatory postsynaptic currents
(AMPAR and NMDAR-eEPSCs) were made from fluorescent transfected neurons and
neighboring untransfected control neurons simultaneously during stimulation of Schaffer
collaterals (Figure 2.5C). This approach permits a pair-wise, internally controlled comparison of
the consequences of the genetic manipulation. As reported previously
56
, we found that expression
of Trio-9 resulted in a selective increase in AMPAR-eEPSC amplitude (Figure 2.5D-E, J-K).
Conversely, we found that knocking down Trio expression in these neurons using a short hairpin
RNA (shRNA) against Trio resulted in a selective reduction in AMPAR-eEPSC amplitude
(Figure 2.5F-G, J-K). Recombinant Trio-9 rescues synaptic phenotypes resulting from Trio
knockdown
56
. We then expressed Trio-9 I1329HLAL* for 6 days in CA1 pyramidal neurons. In
contrast to the increase in AMPAR-eEPSC amplitude we observed with Trio-9, Trio-9
I1329HLAL* resulted in a ~40% decrease in AMPAR-eEPSC amplitude (Figure 2.5H,J). Trio-9
I1329HLAL* expression had no effect on NMDAR-eEPSC amplitude (Figure 2.5I,K). This
phenotype was remarkably similar to that observed with the Trio shRNA. Paired-pulse facilitation
(PPF) was also unchanged by postsynaptic expression of Trio-9 I1329HLAL* indicating that the
expression of this mutant did not affect presynaptic glutamate release (Figure 2.5L).
The selective reduction we observe in AMPAR-eEPSC amplitude produced by Trio-9
I1329HLAL* expression could be due to either a reduction in the number of AMPARs at all
36
glutamatergic synapses or an increase in the number of synapses that lack AMPARs entirely. To
determine which of these possibilities had occurred we performed coefficient of variation (CV)
analysis on eEPSC current amplitudes (Figure 2.5M). CV analysis can be used to determine the
quantal parameters of glutamatergic transmission in control and transfected neurons
149-153
. By
comparing the normalized variance in eEPSC amplitudes from two neurons receiving the same
stimulus, it is possible to determine relative quantal size and quantal content. Changes in quantal
size precisely change both the mean eEPSC and the variance such that the normalized ratio of
mean
2
/variance, also known as coefficient of variation (or CV), remains constant. Changes in
quantal size cause the marker of the mean to fall on the horizontal line seen in Figure 2.5M and in
this case, would denote a change in the number of glutamatergic receptors at each synapse. In
contrast, changes in quantal content will produce proportional changes of equal magnitude in CV
that cause the marker of the mean to fall on the diagonal line and indicate a change in the number
of functional synapses. We observed equal reductions in CV and AMPAR-eEPSC amplitude in
neurons expressing Trio-9 I1329HLAL* (Figure 2.5M). This result suggests a reduction in quantal
content rather than quantal size as responsible for this reduction of AMPAR-eEPSC amplitude.
Given that Trio-9 I1329HLAL* expression did not alter NMDAR-eEPSC amplitude or PPF, this
finding indicates that Trio-9 I1329HLAL* expression increases the number of AMPAR-less or
“silent” synapses. Together these data suggest that Trio-9 I1329HLAL* competes with wild-type
Trio, effectively lowers the concentration of functional Trio at glutamatergic synapses and results
in a selective and potentially pathogenic reduction in synaptic AMPAR function.
Trio-9 K1431M expression inhibits synaptic function
We then examined the Trio missense mutation, Trio-9 K1431M. The individual harboring
this mutation displayed severe autistic symptoms and intellectual disability. It is of note that a
37

38
Figure 2.6
Trio-9 p.K1431M expression reduces the strength of glutamatergic synapses.
A, Predicted alteration to interaction between Trio-9 and Rac1 resulting from Trio-9 K1431M. ID,
intellectual disability. B, Trio-9 K1431M inhibits Trio-9’s ability to activate Rac1. Representative
FLIM color maps of HEK293 cells expressing the Rac1 biosensor alone, the biosensor and Trio-9
K1431M or the biosensor and Trio-9 are shown above. A cropped phasor plot for each condition
is shown below. Dashed ovals identify the lifetime distribution for each condition. C-
D, Scatterplots show eEPSC amplitudes for single pairs of control and transfected neurons (open
circles). Filled circles show mean ± SEM. (Insets) Current traces from control (black) and
transfected (green) neurons (Scale bars: 20 ms for AMPA, 50 ms for NMDA, 20 pA). Bar
graphs show the average eEPSC amplitudes (±SEM) of neurons expressing Trio-9 (in gray, from
Figure 2.5J-K) and Trio-9 K1431M normalized to their respective average control eEPSC
amplitudes. In the bar graphs a Wilcoxon Rank Sum Test was used to compare across independent
conditions (i.e., Trio-9 and Trio-9 K1431M in C, *P < 0.05). c Trio-9 K1431M expression reduced
AMPAR-eEPSC amplitude (n = 10 pairs, *P < 0.05, Wilcoxon Signed Rank Test). D, Trio-9
K1431M expression did not affect NMDAR-eEPSC amplitude (n = 8 pairs, P > 0.05, Wilcoxon
Signed Rank Test). E, Mean ± SEM paired-pulse facilitation (PPF) ratios for Trio-9 K1431M
expressing and paired control neurons (n = 5 pairs, P > 0.05, Student’s t-test). Peak 1-scaled
current traces from control (black) and transfected (green) neurons (Scale bar: 20 ms). n.s., not
significant. F, CV analysis of AMPAR-eEPSCs from pairs of control/Trio-9 K1431M neurons.
CV
−2
graphed against ratio of mean amplitude within each pair (open circles). Filled circle shows
mean ± SEM. (n = 10 pairs)
39

40
Figure 2.7
Trio-9 p.S1575N does not alter Trio-9-mediated potentiation of synaptic AMPAR
expression.
A, Protein domain illustration of Trio-9 S1575N. b-c. Scatterplots to the left show amplitudes of
AMPAR and NMDAR-eEPSCs for single pairs of control and transfected neurons (open circles).
Filled circles show mean ± SEM. (Insets) Sample current traces from control (black) and
transfected (green) neurons (Scale bars: 20 ms for AMPA, 50 ms for NMDA, 20 pA). Bar graphs
show the average eEPSC amplitudes (± SEM) of neurons expressing Trio-9 (from Figure 2.5 J-K)
and Trio-9 S1575N normalized to the corresponding average control neuron eEPSC amplitude. B,
Trio-9 S1575N expression in CA1 pyramidal neurons causes an increase in AMPAR-eEPSC
amplitude relative to untransfected control neurons (n = 7 pairs, *p < 0.05, Wilcoxon Sign Rank
Test). c. Trio-9 S1575N expression in CA1 pyramidal neurons did not affect NMDAR-eEPSC
amplitude (n = 5 pairs, p > 0.05, Wilcoxon Sign Rank Test).

41
previous study using alanine-scanning mutations to identify important residues in Trio’s GEF1
region identified this residue along with R1428 as critical for Rac1 activation
154
. Our modeling of
the K1431M mutation predicted a disruption to a number of hydrogen and water-mediated
interactions resulting in a reduced affinity between Trio’s GEF1/DH1 domain and Rac1
(Figure 2.4 and Table 2.1). FLIM analysis revealed that Trio-9 K1431M, like Trio-9 I1329HLAL*,
severely reduced Trio-9’s ability to activate the Rac1 biosensor in HEK293 cells (Figure 2.6B).
Thus, as predicted, Rac1 activation was severely reduced by this mutation. We then expressed
Trio-9 K1431M in CA1 pyramidal neurons and found that Trio-9 K1431M phenocopied Trio-9
I1329HLAL*. Trio-9 K1431M produced a ~50% reduction in AMPAR-eEPSC amplitude
(Figure 2.6C) that is also likely caused by an increase in silent synapses given that a reduction in
quantal content occurred with no change in NMDAR-eEPSC amplitude or change in PPF
(Figure 2.6D-F). Together these data show that Trio-9 K1431M, like Trio-9 I1329HLAL*, likely
competes with endogenous, wild-type Trio at synapses and results in a reduction in synaptic
AMPAR function. Additionally, we found that a missense mutation in Trio’s GEF1 domain that
was identified in an individual without ASD (Trio-9 S1575N)
147
had no effect on Trio-9’s ability
to increase AMPAR-eEPSC amplitude in CA1 pyramidal neurons (Figure 2.3 and Figure 2.7).
Trio-9 P1461T expression inhibits synaptic function
Additional ASD-related missense mutations predicted to impair the interaction between
Trio’s GEF1/DH1 domain and Rac1 were two mutations in the P1461 position, P1461T and
P1461L (Figure 2.4, Table 2.1 and Figure 2.8A). Both individuals had severe neurodevelopmental
disorders. de novo mutations in the same protein residue position in two unrelated individuals with
similar disorders strongly suggests that such mutations contribute to the development of the
disorder. As both mutations were predicted to destabilize Rac1 binding, we examined the impact
42

43
Figure 2.8
Trio Trio-9 p.P1461T expression reduces the strength of glutamatergic synapses.
A, Predicted alteration to Trio’s GEF1 domain resulting from Trio-9 P1461T. NDD,
neurodevelopmental disorder. B, Trio-9 P1461T inhibits Trio-9’s ability to activate Rac1.
Representative FLIM color maps of HEK293 cells expressing the Rac1 biosensor alone, the
biosensor and Trio-9 P1461T or the biosensor and Trio-9 are shown above. A cropped phasor
plot for each condition is shown below. Dashed ovals identify the lifetime distribution for each
condition. C-D, Scatterplots show eEPSC amplitudes for single pairs of control and transfected
neurons (open circles). Filled circles show mean ± SEM. (Insets) Current traces from control
(black) and transfected (green) neurons (Scale bars: 20 ms for AMPA, 50 ms for NMDA,
20 pA). Bar graphs show the average eEPSC amplitudes (±SEM) of neurons expressing Trio-9
(in gray, from Figure 2.5J-K) and Trio-9 P1461T normalized to their respective average control
eEPSC amplitudes. In the bar graphs a Wilcoxon Rank Sum Test was used to compare across
independent conditions (i.e., Trio-9 and Trio-9 P1461T in C, *P < 0.05). C, Trio-9 P1461T
expression reduced AMPAR-eEPSC amplitude (n = 7 pairs, *P < 0.05, Wilcoxon Signed Rank
Test). D, Trio-9 P1461T expression did not affect NMDAR-eEPSC amplitude (n = 6
pairs, P > 0.05, Wilcoxon Signed Rank Test). E, Mean ± SEM paired-pulse facilitation (PPF)
ratios for Trio-9 P1461T expressing and paired control neurons (n = 5 pairs, P > 0.05, Student’s t-
test). Peak 1-scaled current traces from control (black) and transfected (green) neurons (Scale bar:
20 ms). n.s., not significant. F, CV analysis of AMPAR-eEPSCs from pairs of control/Trio-9
P1461T neurons. CV
−2
graphed against ratio of mean amplitude within each pair (open
circles). Filled circle shows mean ± SEM. (n = 7 pairs)

44
P1461T has on Trio-9 function. While we did observe some FRET in HEK293 cells expressing
Trio-9 P1461T and the Rac1 biosensor, the level of biosensor activation in these cells was lower
than those cells expressing wild-type Trio-9 (Figure 2.8B). Thus, this mutation lowers Trio’s
ability to activate Rac1, albeit not to the extent observed with Trio-9 I1329HLAL* and Trio-9
K1431M. Even though this mutation resulted in a less severe inhibition of Trio-9’s ability to
activate Rac1, expression of Trio-9 P1461T in neurons led to a synaptic phenotype that was similar
to Trio-9 I1329HLAL* and Trio-9 K1431M. Like Trio-9 I1329HLAL* and Trio-9 K1431M, Trio-
9 P1461T expression in hippocampal CA1 pyramidal neurons reduced AMPAR-eEPSC amplitude
(Figure 2.8C) by reducing quantal content without affecting NMDAR-eEPSC amplitude or PPF
(Figure 2.8D-F). Together, these findings again suggest an increase in silent synapse number. Thus,
Trio-9 I1329HLAL*, Trio-9 K1431M and Trio-9 P1461T expression in neurons impact
glutamatergic neurotransmission in a similar manner.
Trio-9 D1368V results in Trio hyperfunction
Lastly, we examined the impact of the GEF1/DH1 D1368V missense mutation found in an
individual with severe intellectual disability. Unlike all the other ASD-related GEF1/DH1
missense mutations that have been identified, the D1368 residue is highly exposed to solvent, and
is located in the GEF1/DH1 subdomain far away from the Rac1 binding interface (Figure 2.4 and
Figure 2.9A). Indeed, our modeling of this mutation did not predict any direct impact on the
stability of Trio’s GEF1 domain or its ability to activate Rac1 (Figure 2.4 and Table 2.1).
Therefore, we were curious if this mutation has any impact on Trio-9 function. We observed higher
levels of Rac1 biosensor activation in cells expressing Trio-9 D1368V compared to wild-type Trio-
9 (Figure 1.2B); this unexpected finding suggested that Trio-9 D1368V increases Trio’s ability to
activate Rac1. Remarkably, we found that expression of Trio-9 D1368V in CA1 pyramidal neurons
45

46
Figure 2.9
Trio-9 p.D1368V produces a hyperfunctional synaptogenic form of Trio.
A, Predicted alteration to Trio’s GEF1 domain produced by Trio-9 D1368V. ID, intellectual
disability. B, Trio-9 D1368V increases Trio-9’s ability to activate Rac1. Representative FLIM
color maps of HEK293 cells expressing the Rac1 biosensor alone, the biosensor and Trio-9
D1368V or the biosensor and Trio-9 are shown above. A cropped phasor plot for each condition
is shown below. Dashed ovals identify the lifetime distribution for each condition. C-
D, Scatterplots show amplitudes of eEPSCs for single pairs of control and transfected neurons
(open circles). Filled circles show mean ± SEM. (Insets) Current traces from control (black) and
transfected (green) neurons (Scale bars: 20 ms for AMPA, 50 ms for NMDA, 20 pA). Bar
graphs show the average eEPSC amplitudes (±SEM) of neurons expressing Trio-9 (in gray, from
Figure 2.5J-K) and Trio-9 D1368V normalized to their respective average control eEPSC
amplitudes. In the bar graphs a Wilcoxon Rank Sum Test was used to compare across independent
conditions (i.e., Trio-9 and Trio-9 D1368V in C-D , *P < 0.05). C, Trio-9 D1368V expression
increased AMPAR-eEPSC amplitude (*P < 0.05, Wilcoxon Signed Rank Test). D, Trio-9 D1368V
expression increased NMDAR-eEPSC amplitude (n = 6 pairs, *P < 0.05, Wilcoxon Signed Rank
Test). E, Mean ± SEM paired-pulse facilitation (PPF) ratios for Trio-9 D1368V expressing and
paired control neurons (P > 0.05, Student’s t-test). Peak 1-scaled current traces from control (black)
and transfected (green) neurons (Scale bar: 20 ms). n.s., not significant. F, CV analysis of AMPAR
and NMDAR-eEPSCs from pairs of control/Trio-9 D1368V neurons. CV
−2
graphed against ratio
of mean amplitude within each pair (open circles). Filled circle shows mean ± SEM. (AMPAR-
eEPSCs, n = 6 pairs; NMDAR-eEPSCs, n = 6 pairs). G, Trio-9 D1368V expression increased
dendritic spine density. Representative dendritic spine images from neurons transfected with GFP
47
(control), wild-type Trio-9 or Trio-9 D1368V are shown above (Scale bars: 1 μm). The bar
graph below shows average spine density (mean ± SEM) of neurons expressing wild-type Trio-9
or Trio-9 D1368V normalized and compared to GFP expressing control neurons (control, n = 6;
Trio-9, n = 5; Trio-9 D1368V, n = 5, *P < 0.05, Student’s t-test)

48
resulted in a dramatic increase in AMPAR-eEPSC amplitude that was about twice that seen with
wild-type Trio-9 (Figure 2.9C). Trio-9 D1368V expression in CA1 pyramidal neurons also led to
an unexpected increase in NMDAR-eEPSC amplitude (Figure 2.9D). PPF was not affected by this
mutation (Figure 2.9E). To determine whether the observed enhancements of AMPAR and
NMDAR-mediated transmission were produced by an increase in the number of glutamatergic
synapses or an increase in the expression of AMPARs and NMDARs at existing synapses we again
performed CV analysis. We found that increases in both AMPAR and NMDAR-eEPSC amplitude
likely result from changes in quantal content rather than quantal size (Figure 2.9F). This finding
suggested that Trio-9 D1368V-mediated enhancement of AMPAR and NMDAR function is
produced by an increase in the number of functional glutamatergic synapses. We reasoned that if
Trio-9 D1368V expression leads to the formation of additional glutamatergic synapses, then a
corresponding increase in dendritic spine density should be observed in these neurons. We used
Structured Illumination Microscopy (SIM) to obtain super resolution images of dendritic spines
from neurons expressing GFP, wild-type Trio-9 or Trio-9 D1368V. While wild-type Trio-9
expression had no effect on dendritic spine density, expression of Trio-9 D1368V resulted in a
greater than twofold increase in the density of dendritic spines compared to GFP expressing control
neurons (Figure 2.9G). Together these data reveal that the Trio-9 D1368V mutation is a
hypermorphic mutation resulting in a hyperfunctioning synaptogenic form of Trio that when
expressed in neurons is capable of producing an abnormally high number of glutamatergic
synapses.
1.5 Discussion
Here we find a hotspot of de novo ASD-related mutations clustered specifically in Trio’s
GEF1/DH1 subdomain (P-value < 5.5 × 10
−13
). Such a high number of potentially function-
49
disrupting mutations suggests that these mutations contribute to the development of ASD-related
disorders. In support of this hypothesis, we found that every GEF1/DH1 ASD-related mutation we
tested greatly altered Trio’s influence on glutamatergic synapse function in the hippocampus. We
identify both hypomorphic and hypermorphic ASD-related mutations in Trio, thus implicating
reduced as well as excessive Trio activity in ASD-related pathogenesis. Trio’s involvement in
ASD-related disease is further reinforced by recent human genome analysis studies that rank TRIO
among their top ASD-candidate genes
155
.
Specific association of Trio mutations with ASD is supported by analysis of its close
paralog, Kalirin, which has a unique developmental expression profile. No ASD-associated
missense mutations were observed in Kalirin, despite playing a similar role in glutamatergic
synapse regulation
56
. Trio is highly expressed in early postnatal development and decreases with
age
123
. In contrast, Kalirin expression does not peak until adolescence
124,125
. Remarkably, Kalirin
function has been implicated in later onset neuropsychiatric disorders like schizophrenia and
Alzheimer’s disease
156-159
. A missense mutation in Kalirin’s GEF1/DH1 domain that inhibited
Rac1 activation has also been found in an individual with schizophrenia
157
. Thus, the expression
profiles of Trio and Kalirin appear to match the age of onset of the diseases in which they are
implicated, and suggest that disruption of Rac1-mediated synaptic regulation at different time
points of brain development gives rise to distinct brain-related diseases.
Dysregulation of glutamatergic transmission is believed to underlie the development of
ASD, and existing animal models of ASD display varied glutamatergic synapse
phenotypes
160
. MeCP2 Tg1 and MET knockout mice, for example, display enhanced
glutamatergic neurotransmission while SHANK3 knockout and MeCP2 null/y mice display
deficits in glutamatergic signaling
70,129,130
. While the mechanisms underlying the glutamatergic
50
synapse phenotypes observed in these animals are unclear, such data suggest that alteration of
glutamatergic synaptic strength in either direction during synapse formation in early postnatal
development likely results in global network changes contributing to ASD-related disease. By
regulating actin filament formation through its GEF1 domain, Trio regulates glutamatergic
synapse function via its effect on synaptic structure. Here we find that ASD-related missense
mutations in Trio give rise to both decreased and increased glutamatergic synaptic function
resulting from an increase in silent synapse number or enhanced synaptogenic capability,
respectively. Thus, our data suggest that pathological repercussions result from both Trio
hypofunction and Trio hyperfunction during development. Furthermore, to the best of our
knowledge, our study is the first to show that ASD-related missense mutations in a single protein
can produce bidirectional alterations of glutamatergic synapse function.
Our modeling and FLIM analysis identify the majority of Trio’s ASD-related GEF1/DH1
mutations as detrimental to Trio’s ability to activate Rac1. We also find that expression of several
hypomorphic ASD-related Trio mutants in CA1 pyramidal neurons leads to selective reductions
in AMPAR-eEPSC amplitude. While the exact mechanism of ASD-related hypomorphic
mutations reducing synaptic AMPAR expression is unknown, the likely explanation is that the
expressed hypomorphic Trio mutants compete with wild-type Trio for access to synapses and
effectively lower the synaptic concentration of functional Trio. It is additionally possible that these
hypomorphic Trio proteins that lack functional GEF1 domains compete with wild-type Trio for
association with key synaptic regulatory protein complexes. Alternatively, expressed hypomorphic
Trio mutants may target and promote degradation of wild-type Trio through some unknown
mechanism. Such antagonistic relationships between hypomorphic and wild-type forms of Trio
are important because the individuals that harbor the de novo Trio mutations described in this
51
study are heterozygous for these mutations. Given that the majority of ASD-related mutations in
Trio’s GEF1/DH1 domain potentially work against wild-type Trio function, these individuals are
expected to have a level of Trio function below what would occur with a simple deletion of one
copy of the gene. Alternatively, it is possible that these ASD-related Trio mutations are expressed
at lower levels compared to wild-type Trio or are mis-targeted and thus do not reach synapses.
However, we would presume that if these were the primary effects of these mutations,
overexpression of such mutants would produce either smaller increases in AMPAR-eEPSC
amplitude or simply produce no effect on synaptic transmission relative to control neurons. What
we observe with hypomorphic Trio mutant expression are similar reductions in AMPAR-eEPSC
amplitude that are consistent with these mutants inhibiting wild-type Trio function through either
competition or degradation. Going forward, knock-in mice heterozygous for these hypomorphic
ASD-related Trio mutations will be necessary to precisely identify ASD-related behavioral
phenotypes that arise when Trio function is potentially in-between what occurs in Trio knockout
mice and mice with a single TRIO allele. These mice will also be very useful in examining the
impact ASD-related Trio mutations have on synaptic function at different time points in
development, and may aid in identifying novel ways in which these mutations impact
glutamatergic synapse function. For example, Trio’s recent implication in neurotransmitter release
at the Drosophila neuromuscular junction now justifies an investigation into possible presynaptic
roles for Trio at mammalian synapses
161
.
In this study, we also identify a hypermorphic missense mutation in Trio’s GEF1 domain
that produces a dramatic enhancement of Trio activity. We find that Trio-9 D1368V expression
results in an enhancement of AMPAR-mediated transmission that is twice that produced by wild-
type Trio. Trio-9 D1368V also results in a substantial enhancement of NMDAR-mediated currents,
52
which are not affected by wild-type Trio. CV and dendritic spine analysis identify an increase in
the number of functional synapses as being responsible for this observed increase in glutamatergic
neurotransmission. This finding is akin to reports of increased glutamatergic neurotransmission in
several animal models of ASD/ID and observed increases in the number of glutamatergic synapses
in a number of individuals with ASD
129,130,162
. Interestingly, this mutation is the only ASD-related
mutation identified in our study that was predicted to not directly influence Trio’s ability to activate
Rac1. Furthermore, this residue is highly exposed to solvent on a region of the GEF1/DH1
subdomain that is pointing away from the interface with Rac1. One possibility is that D1368V
destabilizes autoinhibitory interactions that occur in Trio. Such intramolecular interactions have
been proposed for Kalirin and other RhoGEFs
163,164
. Another possibility is that this site is involved
in the binding of allosteric inhibitors of GEF domains. TRIOBP, for example, has been shown to
bind directly to Trio’s GEF1 domain and inhibit the ability of Trio to activate Rac1
165
.
Alternatively, it is possible that this mutation increases Trio expression levels (e.g., through
disruption of an E3 ligase binding substrate). Additional study will be needed to elucidate the
mechanism by which this mutation results in enhanced Trio function.
Disruptions in synaptic Rac1-mediated actin regulation have recently been found to
underlie the development of ASD-related behavioral phenotypes in well-established animal
models of ASD-related diseases
116,118,119
. A recent study also identified Rac1 as a likely point of
convergence of a number of ASD risk genes
117
. Here we identify the Rac1 activation domain of
the synaptic actin regulatory protein, Trio, as a hotspot for ASD-related de novo mutations.
Because dysregulation of glutamatergic neurotransmission is believed to underlie the behavioral
phenotypes in many animal models of ASD and because proteins downstream of Trio activity have
not been found to harbor substantial ASD-related mutations, it is tempting to speculate that altered
53
Trio function represents a major point of convergence of a number of factors that give rise to ASD.
Going forward it will be necessary to determine the prevalence of altered Trio function in
individuals with ASD and establish whether Trio-related therapies can be applied to reverse ASD-
related behavioral phenotypes in appreciable numbers of individuals with this disease.

54
Chapter 2: An Intellectual Disability-Related Missense Mutation in Rac1
Prevents LTP Induction
2.1 Abstract
The small GTPase Rac1 promotes actin polymerization and plays a critical and
increasingly appreciated role in the development and plasticity of glutamatergic synapses.
Growing evidence suggests that disruption of the Rac1 signaling pathway at glutamatergic
synapses contributes to Autism Spectrum Disorder/intellectual disability (ASD/ID)-related
behaviors seen in animal models of ASD/ID. Rac1 has also been proposed as a strong candidate
of convergence for many factors implicated in the development of ASD/ID. However, the effects
of ASD/ID-related mutations in Rac1 itself have not been explored in neurons. Here, we
investigate a recently reported de novo missense mutation in Rac1 found in an individual with
severe ID. Our modeling predicts that this mutation will strongly inhibit Rac1 activation by
occluding Rac1’s GTP binding pocket. Indeed, we find that this de novo mutation prevents Rac1
function and results in a selective reduction in synaptic AMPA receptor function. Furthermore,
this mutation prevents the induction of long-term potentiation (LTP), the cellular mechanism
underlying learning and memory formation. Together, our findings strongly suggest that this
mutation contributes to the development of ID in this individual. This research demonstrates the
importance of Rac1 in synaptic function and plasticity and contributes to a growing body of
evidence pointing to dysregulation of actin polymerization at glutamatergic synapses as a
contributing factor to ASD/ID.
2.2 Introduction
Intellectual disability (ID) is a neurodevelopmental disorder defined by significant
limitations in intellectual functioning and adaptive behavior and is often comorbid with autism
55
spectrum disorders(ASD)
166
. Human and animal ASD/ID model research has converged on altered
glutamatergic synaptic function as a potential cause of cognitive dysfunction
167-169
. Synapses allow
communication between neurons and are essential for learning and memory formation in the brain.
Learning and memory formation rely on long lasting increases in glutamatergic synapse strength
produced by the cellular process of long-term potentiation (LTP). Synaptic strength is largely
influenced by changes in synaptic structure
35,170
, and synaptic structure is dictated by regulation of
the synaptic actin-cytoskeleton. Rho GTPases are key regulators of actin polymerization and have
been generally implicated in ASD/ID
171
. Disruption of the small Rho GTPase Rac1, in particular,
has been identified in common ASD/ID animal models, and Rac1 has been proposed as a
convergence point for many ASD/ID genes
117-119,122,172
.
Rac1 orchestrates synaptic actin polymerization and is essential for synaptic function and
plasticity
173-175
. Rac1 switches between an active GTP-bound state and an inactive GDP-bound
state, and Rac1’s activity is tightly regulated by protein activators (GEFs) and inhibitors (GAPs).
Guanine nucleotide exchange factors, or GEF proteins, activate Rac1 by exchanging GDP for GTP
while GTPase accelerating proteins, or GAP proteins, induce GTP hydrolysis. In a previous study,
we discovered that Rac1 GEFs, Kalirin and Trio, are essential for the induction of LTP
56
.
Furthermore, we have found an ASD-related de novo mutation hotspot in Trio’s Rac1 activating
domain. These ASD-related mutations in Trio bidirectionally alter Rac1 activation, leading to
either abnormally weak or strong glutamatergic synapses
122
. However, the influence of ASD/ID
related mutations in Rac1 itself has not been explored in neurons.
Recently, analysis of 2104 patient-parent trios among multiple ID studies identified 10 new
ID related genes
176
. Among these genes was RAC1. In this exome sequencing study, an individual
with severe ID was found harboring a de novo mutation in the RAC1 gene that changes the
56
cysteine in position 18 of the Rac1 protein (C18) to a tyrosine (C18Y; Figure 2.1). Here, we
characterize the impact of this recently reported Rac1 ID-related de novo missense C18Y mutation
on synaptic function. Computational modeling, electrophysiological recording and super
resolution imaging demonstrate that this severe ID-related mutation occludes Rac1’s GTP binding
pocket and disrupts the function of glutamatergic synapses. Furthermore, we find that the
expression of this severe ID-related Rac1 mutation blocks LTP, the cellular basis of learning and
memory formation. Our results suggest that the ID observed in this individual stems from altered
Rac1 function and the resulting synaptic dysfunction. This study supports synaptic Rac1-mediated
actin regulation as a key convergence point of molecular perturbations previously associated with
ASD/ID.
2.3 Materials and Methods
Electrophysiology: organotypic hippocampal slices
This study was carried out in accordance with the National Institutes of Health (NIH) Guide
for the Care and Use of Laboratory Animals and the protocol was approved by the University of
Southern California Institutional Animal Care and Use Committee. 400 μm organotypic
hippocampal slice cultures were prepared from P6 to P9 Sprague-Dawley rat pups as described
previously
138
. Culture media was exchanged every other day. Sparse biolistic transfections of
organotypic slice cultures were carried out on DIV4 or DIV12 as described
139
. Construct
expression was confirmed by GFP and mCherry co-transfection. Paired whole-cell recordings
from transfected neurons and non-transfected control neurons were performed on DIV7 or DIV15
slices. During recording, all slices were maintained in room temperature artificial cerebrospinal
fluid (aCSF) saturated with 95% O2/5% CO2. Whole-cell recordings were carried out as
described
122
. Paired-pulse ratio was recorded by delivering two stimuli at intervals of 20 ms, 40
57
ms, 70 ms and 100 ms and dividing the peak response of stimulus two by the peak response of
stimulus one. 0.1 mM spermine was added to intracellular 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. This calculation was as follows: RI
= 7(I40 – I0)/4(I0 – I70) where Ix represent EPSC amplitude at x mV. A built-in single
exponential decay fit function in IgoR was used to calculate decay time constants for AMPAR-
eEPSCs. No more than one paired recording was performed on a given slice.
Electrophysiology: acute hippocampal slices
Mice were electroporated on E15 as previously described
56
. 300 μm acute hippocampal
slices from P21 to P29 mice were prepared using D.S.K microslicer ZERO1 vibrating microtome
(Ted Pella, CA, USA) in high sucrose low sodium ice-cold cutting solution saturated with 95%
O2/5% CO2. Cutting solution contained 2.5 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, 1.25 mM
NaH2PO4, 25 mM NaHCO3, 7 mM glucose, 210 mM sucrose and 1.3 mM ascorbic acid. After
cutting, slices were incubated in aCSF at 37°C for at least 40 mins and at room temperature for
another 40 mins before recording.
Slices were transferred to a submersion chamber for recording and maintained in room
temperature aCSF saturated with 95% O2/5% CO2. Synaptic responses were evoked by
stimulating with a monopolar glass electrode filled with aCSF in stratum radiatum of CA1.
AMPAR currents were measured at −70 mV. A “pairing” stimulation protocol was used to induce
LTP. Our pairing LTP induction protocol consisted of a single train of 2 Hz Schaffer collateral
stimulation for 90 s while holding the postsynaptic neuron at 0 mV. This induction protocol was
applied within 5 mins of achieving whole-cell configuration to avoid “wash-out” of LTP.
58
Individual experiments were normalized to the baseline before stimulation and 12 consecutive
responses were averaged to generate 1-min bins, which were then averaged to generate summary
graphs. Bar graphs of LTP magnitudes were produced based on the averaged eEPSC values for
the first 2 mins (prior to LTP induction) and last 2 mins of LTP summary graphs.
Coefficient of variation analysis
The locus of alterations of eEPSC amplitude was estimated by comparing the change in
eEPSC variance with the change in mean amplitude
149,151,152
. The coefficient of variation (CV)
was calculated as SD/M, where M and SD are the mean and standard deviation of eEPSC
amplitude, respectively. The M and SD were measured for a concurrent set of stimuli (25–60
sweeps per pair) from a control and neighboring transfected cell. Pairs with less than 25 sweeps
were excluded from CV analysis. It has been shown 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 × presynaptic release probability, Pr; CV
−2
= nPr/(1 −
Pr)
149,150,152,177
. 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 suggest changes in quantal content while values approaching
the horizontal line (y = 1) suggest a change in quantal size. Unsilencing of synapses can mimic an
increase in the number of release sites when presynaptic release probability is unchanged
178
. Linear
regressions were obtained using the least squares method.
Spine density analysis
Control and experimental CA1 pyramidal neurons in cultured hippocampal slice prepared
from P6 to P8 rat pups were biolistically transfected with pFUGW-GFP and pCAGGS-IRES-
59
mCherry constructs on DIV4. Images were acquired by experimenter, blinded to condition, on
DIV7 using super-resolution microscopy (Elyra Microscope System, Zeiss). For use with the
inverted microscope and oil-immersion 100× objective lens, slices were fixed in 4% PFA/4%
sucrose in PBS, washed 3× with PBS and cleared with an abbreviated SeeDB-based protocol
140
.
Image acquirement and analysis were carried out as described previously
122
.
Modeling
The effect of mutations on GTP binding were predicted using the high-resolution structure
of Rac1 in complex with the slowly hydrolyzing GTP analog guanosine-5′-(βγ-imido) triphosphate
(GMP-PNP; PDB code 3TH5). Calculations were performed using ICM molecular modeling
software (Molsoft LLC).
Experiment Constructs
Human Rac1 and Rac1b cDNA were purchased from Genescript (clone ID OHu23004D
for Rac1 and OHu22224C for Rac1b) and cloned into a pCAGGS vector containing IRES-
mCherry. ID-related mutations were made from Rac1 and Rac1b cDNA using overlap-extension
PCR followed by in-fusion cloning (Clontech). All plasmids were confirmed by DNA sequencing.
A pFUGW vector expressing only GFP was co-expressed with pCAGGS-IRES-mCherry
constructs to enhance identification of transfected neurons and was used as a control vector for
spine imaging.
2.4 Results
A severe ID-related de novo mutation is predicted to prevent Rac1 activation
A recent exome sequencing study identified RAC1 as a novel ID risk gene
176
. This study
identified an individual with severe ID that harbored a de novo missense mutation resulting in the
substitution of a cysteine residue for a tyrosine at position 18 (C18Y) of Rac1’s amino acid
60

Figure 3.1
A de novo missense mutation in the P-loop region of Rac1 in an individual with severe
intellectual disability (ID) is predicted to prevent Rac1 activation.
(Top) Rac1 protein regions are indicated, starting with the N terminus: P-loop region, switch I and
switch II. Location of a de novo ID-related missense mutation and control missense mutations are
shown. (Bottom) The predicted effect of the severe ID-related mutation is shown. Rac1’s ribbon
structure is shown in orange (Protein Data Bank code 3TH5). Rac1’s GTP binding pocket is shown
as a transparent white surface. The tyrosine at position 18 observed in an individual with severe
ID is labeled and shown in stick representation and colored magenta. A GTP analog (GMP-PNP)
is shown in stick representation with carbon atoms colored pale yellow.

61
sequence. This residue is inside the P-loop region of Rac1. Specifically, this residue resides within
Rac1’s GTP binding pocket. Previous structural analysis suggests that Rac1 C18 interacts directly
with GTP
179,180
. Together, this evidence suggests that C18 plays a role in Rac1 function. We found
no mutations in Rac1’s P-loop in the ExAC control genome database (Figure 3.1)
146
, which
accentuated the importance of residue C18. We then used a structure-based modeling approach to
predict the effect of this ID-related mutation on Rac1 function. Our modeling of the C18Y mutation
showed that replacing the cysteine at this position with a tyrosine causes the aromatic R group of
the tyrosine residue to extend into the middle of the Rac1’s GTP binding pocket. Thus, this ID-
related mutation is likely to disrupt GTP-mediated activation of Rac1 (Figure 3.1).
Rac1 C18Y inhibits synaptic function
Regulation of glutamatergic synapse strength is critical for information storage in the brain,
and Rac1 is a key regulator of glutamatergic synapses. To examine the impact of this ID-related
Rac1 mutation on glutamatergic neurotransmission, we used biolistic transfection to express wild-
type Rac1 or Rac1 C18Y in CA1 pyramidal neurons of rat organotypic hippocampal slice cultures.
Given that ID is a neurodevelopmental disorder, we reasoned that Rac1 C18Y may impact synaptic
development. To test this, we transfected CA1 pyramidal neurons in DIV4 hippocampal cultures
with either Rac1 or Rac1 C18Y constructs. Three days after transfection, we recorded AMPA
receptor and NMDA receptor-evoked excitatory postsynaptic currents (AMPAR and NMDAR-
eEPSCs) following Schaffer collateral stimulation from transfected fluorescent neurons and
neighboring untransfected control neurons, simultaneously (Figure 3.2A-B). This approach
permits a pair-wise, internally controlled comparison of the consequences of the genetic
manipulation. We found that Rac1 overexpression produced a 2-fold increase in AMPAR-eEPSC
amplitude (Figure 3.2C). In marked contrast to wild-type Rac1, expression of Rac1 C18Y led to a
62

63
Figure 3.2
Rac1 C18Y weakens glutamatergic synaptic transmission.
A, Electrophysiology recording setup. B, Timeline of transfection and recording. C-
D, Scatterplots show eEPSC amplitudes recorded at DIV7 for single pairs of CA1 pyramidal
neurons transfected with Rac1 (green) or Rac1 C18Y (red) and their corresponding control neurons
(open circles). Filled circles show mean ± SEM (insets). Current traces from control (black) and
transfected (green for Rac1, red for Rac1 C18Y) neurons are shown (Scale bars: 20 ms for AMPA,
50 ms for NMDA, 20 pA). Bar graphs show the average eEPSC amplitudes (± SEM) of neurons
expressing Rac1 or Rac1 C18Y normalized to their respective average control eEPSC amplitudes
at DIV7 and DIV15. Wilcoxon Rank Sum Tests were used to compare across independent
conditions (i.e., Rac1 and Rac1 C18Y in C, *P < 0.05). C, Rac1 expression increased AMPAR-
eEPSC amplitude in DIV7 (n = 8 pairs, *P < 0.05) and DIV15 slices (n = 9 pairs, *P < 0.05). Rac1
C18Y expression reduced AMPAR-eEPSC amplitude in DIV7 (n = 9 pairs, *P < 0.05) and DIV15
slices (n = 7 pairs, *P < 0.05). Significance was determined by Wilcoxon Signed Rank
Tests. D, Rac1 and Rac1 C18Y expression did not affect NMDAR-eEPSC amplitude in DIV7
(Rac1: n = 9 pairs, P > 0.05, Rac1 C18Y: n = 12 pairs, P > 0.05, Wilcoxon Signed Rank Test) or
in DIV15 slices (Rac1: n = 9 pairs, P > 0.05, Rac1 C18Y: n = 7 pairs, P > 0.05, Wilcoxon Signed
Rank Test). n.s., not significant. E, Rac1 and Rac1 C18Y expression did not affect paired-pulse
facilitation (PPF) ratios at interstimulus intervals of 20, 40, 70 and 100 ms (Left plot: 20 ms: n =
7 pairs, 40 ms: n = 5 pairs, 70 and 100 ms: n = 6 pairs, P > 0.05, Student’s t-test; Right plot: 20
ms: n = 7 pairs, 40 ms: n = 8 pairs, 70 ms: n = 5 pairs, 100 ms: n = 6 pairs, P > 0.05, Student’s t-
test). Peak 1-scaled current traces from control (black) and transfected (green for Rac1, red for
Rac1 C18Y) neurons are shown. (Scale bar: 20 ms). n.s., not significant. F, Rac1 and Rac1 C18Y
64
expression did not change AMPAR-eEPSC rectification. Bar graphs show mean ± SEM of the
AMPAR-eEPSC rectification index recorded in the presence of AP5. (Left graph: control: n = 5,
Rac1: n = 5, p > 0.05, Wilcoxon Signed Rank Test; Right graph: control: n = 7, Rac1 C18Y: n =
6, p > 0.05, Wilcoxon Signed Rank Test). Representative traces (green for Rac1, red for Rac1
C18Y) are shown to the left of graph (Scale bars: 20 ms). G, Rac1 and Rac1 C18Y expression did
not affect AMPAR-eEPSC decay. Bar graphs show mean ± SEM of the AMPAR-eEPSC decay
kinetics. (Left graph: n = 8, p > 0.05, Wilcoxon Signed Rank Test; Right graph: n = 7, p > 0.05,
Wilcoxon Signed Rank Test). Representative traces (green for Rac1, red for Rac1 C18Y) are
shown to the left of graphs (Scale bars: 20 ms). H, Coefficient of variation (CV) analysis of
AMPAR-eEPSCs from pairs of control and Rac1/Rac1 C18Y neurons at DIV7 and DIV15.
CV
−2
ratios are graphed against the mean amplitude ratio for each pair (open circles; green for
Rac1 DIV7: n = 7 pairs; blue for Rac1 DIV15: n = 9 pairs; red for Rac1 C18Y DIV7: n = 5
pairs; yellow for Rac1 C18Y DIV15: n = 7 pairs). Filled circles show mean ± SEM. Dashed lines
show linear regression and 95% confidence intervals. I, Rac1 and Rac1 C18Y expression did not
affect spine density. Representative dendritic spine images from neurons transfected with GFP
(control), Rac1 or Rac1b C18Y are shown on the left (Scale bars: 5 μm). The bar graph shows
average spine density (mean ± SEM) of neurons expressing Rac1 or Rac1 C18Y normalized to
GFP expressing control neurons (control: 0.28 ± 0.024 spines/μM, n = 8, Rac1: 0.31 ± 0.024
spines/μM, n = 11, Rac1 C18Y: 0.26 ± 0.021 spines/μM, n = 16, P > 0.05, Student’s t-test).

65
60% decrease in AMPAR-eEPSC amplitude (Figure 3.2C). The alterations of synaptic function
produced by Rac1 and Rac1 C18Y were selective for AMPAR-eEPSC amplitude, with no change
observed in NMDAR-eEPSC amplitudes (Figure 3.2D). Together, these data demonstrate Rac1’s
effect on glutamatergic synapses is severely altered by the C18Y mutation, and that Rac1 C18Y
produces an inhibitory effect on AMPAR-mediated synaptic transmission. To determine if specific
time points in development are sensitive to Rac1 C18Y expression, we expressed either Rac1 or
Rac1 C18Y in older CA1 pyramidal neurons in DIV12 cultures and recorded from these neurons
3 days later at DIV15 (Figure 3.2B). Expression of Rac1 and Rac1 C18Y in DIV15 cultures
produced effects on synaptic transmission that were very similar to DIV7 cultures (Figures 3.2C,
D). We then examined whether the alterations in AMPAR-eEPSC amplitude we observed occur
as a result of changes in presynaptic glutamate release by evaluating paired-pulse facilitation (PPF).
We found that neither neurons transfected with Rac1 nor neurons transfected with Rac1 C18Y
exhibited any change in PPF compared to control neurons over a range of paired stimulation
intervals. These data demonstrate that Rac1 and Rac1 C18Y-mediated alterations in AMPAR-
eEPSC amplitude do not result from alterations in presynaptic glutamate release (Figure 3.2E).
Given that AMPAR subunit composition affects AMPAR function, we asked whether
subunit composition of postsynaptic AMPARs is altered by either overexpression of wild-type
Rac1 or expression of Rac1 C18Y. AMPARs are composed of heterotetrameric assemblies of
GluA1–4 subunits. In CA1 pyramidal neurons ~80% of synaptic AMPARs are GluA1/A2
heteromers and the remaining ~20% are GluA2/A3 heteromers
181
. GluA2-containing AMPARs
exhibit a near linear I/V relationship whereas AMPARs lacking GluA2 are Ca2+ permeable and
display marked inward rectification. We performed rectification assays to determine if our genetic
manipulations produced insertion of GluA2-lacking AMPARs into CA3-CA1 synapses. Neither
66
Rac1 overexpression nor Rac1 C18Y expression altered AMPAR-eEPSC rectification, and thus
these genetic manipulations do not alter the GluA2 content of synaptic AMPARs (Figure 3.2F).
Next, we wanted to determine if Rac1 overexpression or Rac1 C18Y expression alter the
GluA1/A2 to GluA2/A3 AMPAR ratio at glutamatergic synapses. GluA1/A2 AMPARs exhibit
slower decay kinetics relative to GluA2/A3 AMPARs, thus a shift in the GluA1/A2 to GluA2/A3
AMPAR ratio would manifest as a change in AMPAR-eEPSC decay kinetics
182
. Both Rac1 and
Rac1 C18Y transfected neurons showed the same decay kinetics as that of control neurons
(Figure 3.2G). Thus, synaptic AMPAR subunit composition does not change in response to our
genetic manipulations.
The selective alterations in AMPAR-eEPSC amplitudes produced by the overexpression
of Rac1 or Rac1 C18Y expression could be due to a change in the relative number of AMPAR
containing or AMPAR-lacking (“silent”) glutamatergic synapses (i.e., a change in quantal content).
Alternatively, alterations in AMPAR-eEPSC amplitude could be due to a global change in the
efficiency of AMPAR-mediated synaptic transmission across all functional synapses (a change in
quantal size). To answer this question we used coefficient of variation analysis (CV analysis). 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 (Figure 3.2H). It has been shown theoretically and experimentally that changes in CV value
are independent of quantal size but vary in a predictable manner with quantal content
149,150,152
.
Thus, values approaching the horizontal line (y = 1) indicate a change in quantal size. In contrast,
values close to the diagonal (y = x) line indicate a change in quantal content
151,153
. Our CV analysis
of Rac1 overexpression resulted in a linear regression line of the data falling closer to the horizontal
line (Figure 3.2H). 95% confidence intervals of the data include the horizontal line but do not
67
include the diagonal line. Therefore, we conclude that Rac1 overexpression-mediated increases in
AMPAR-eEPSC amplitude largely arise from a uniform increase in AMPAR-mediated
neurotransmission efficiency across all functional synapses. Interestingly, CV analysis of Rac1
C18Y data yielded average points that fall on the diagonal line and 95% confidence intervals that
include the diagonal line and exclude the horizontal line (Figure 3.2H). These data indicate that
Rac1 C18Y results in a reduction in the number of synapses that contain functional AMPARs.
This could be due to reduction of synapse number or increase of AMPAR lacking synapses. We
then used Structural Illumination Microscopy (SIM) to obtain super resolution images of dendritic
spines from CA1 pyramidal neurons expressing GFP alone, GFP and Rac1 or GFP and Rac1 C18Y.
We found neither Rac1 overexpression nor Rac1 C18Y expression resulted in a change in dendritic
spine density (Figure 3.2I). Given that Rac1 C18Y expression does not affect NMDAR-eEPSC
amplitude or PPF, these data suggest that Rac1 C18Y expression results in a potentially pathogenic
increase in the number of AMPAR-lacking or “silent” glutamatergic synapses.
Rac1 C18Y prevents GFP-mediated activation of Rac1
Our modeling suggests that Rac1 C18Y will prevent GTP from activating Rac1. GTP
associates with Rac1 resulting in Rac1 activation. Rac1 is able to inactivate itself through its ability
to hydrolyze bound GTP, converting it to GDP. Guanine nucleotide exchange factors (GEFs)
regulate Rac1 function by binding to Rac1 and removing GDP, allowing Rac1 to reassociate with
GTP and reactivate. Thus, preventing Rac1’s ability to convert GTP to GDP renders Rac1
constitutively active. The only way to prevent activation of a constitutively active form of Rac1 is
to directly interfere with GTP’s interaction with Rac1. If the C18Y mutation inhibits Rac1b
function, this result will point to C18Y directly preventing a functional GTP interaction with Rac1.
68

69
Figure 3.3
The ID-related mutation C18Y prevents synaptic potentiation by a constitutively active form
of Rac1, Rac1b.
A-B, Scatterplots show eEPSC amplitudes for single pairs of CA1 pyramidal neurons transfected
with Rac1b (green) or Rac1b C18Y (red) and their corresponding control neurons (open circles).
Filled circles show mean ± SEM (insets). Current traces from control (black) and
transfected (green for Rac1b, red for Rac1b C18Y) neurons are shown (Scale bars: 20 ms for
AMPA, 50 ms for NMDA, 20 pA). Bar graphs show the average eEPSC amplitudes (± SEM) of
neurons expressing Rac1b or Rac1b C18Y normalized to their respective average control eEPSC
amplitudes. Wilcoxon Rank Sum Tests were used to compare across independent conditions (i.e.,
Rac1b and Rac1b C18Y in A-B, *P < 0.05). A, Rac1b expression increased AMPAR-eEPSC
amplitude (n = 12 pairs, *P < 0.05, Wilcoxon Signed Rank Test). Rac1b C18Y expression reduced
AMPAR-eEPSC amplitude (n = 9 pairs, *P < 0.05, Wilcoxon Signed Rank Test). B, Rac1b
expression increased NMDAR-eEPSC amplitude (n = 10 pairs, *P < 0.05, Wilcoxon Signed Rank
Test). Rac1b C18Y expression did not affect NMDAR-eEPSC amplitude (n = 8 pairs, P > 0.05,
Wilcoxon Signed Rank Test). C, Rac1b and Rac1b C18Y expression did not affect PPF ratios at
interstimulus intervals of 20, 40, 70 and 100 ms (Upper plot: 20 ms: n = 6 pairs, 40 ms: n = 8 pairs,
70 ms: n = 4 pairs, 100 ms: n = 6 pairs, P > 0.05, Student’s t-test; Lower plot: 20 ms: n = 6 pairs,
40 ms: n = 8 pairs, 70 ms: n = 5 pairs, 100 ms: n = 6 pairs, P > 0.05, Student’s t-test). Peak 1-
scaled current traces from control (black) and transfected (green for Rac1b, red for Rac1b C18Y)
neurons are shown. (Scale bar: 20 ms). n.s., not significant. D, CV analysis of AMPAR-eEPSC
from pairs of control and Rac1b/Rac1b C18Y neurons and of NMDAR-eEPSCs from pairs of
control and Rac1b neurons. CV
−2
ratios are graphed against the mean amplitude ratio for each pair
70
(open circles) (green for Rac1b AMPAR-eEPSC: n = 12 pairs; black for Rac1b NMDAR-
eEPSC: n = 10 pairs; red for Rac1b C18Y AMPAR-eEPSC: n = 9 pairs). Filled circles show mean
± SEM. Dashed lines show linear regression and 95% confidence intervals. E, Rac1b expression
but not Rac1b C18Y expression increased dendritic spine density. Representative dendritic spine
images from neurons transfected with GFP (control), Rac1b, or Rac1b C18Y are shown on the left
(Scale bars: 5 μm). The bar graph shows average spine density (mean ± SEM) of neurons
expressing Rac1b (control: 0.17 ± 0.015 spines/μM, n = 11; Rac1b: 0.31 ± 0.031 spines/μM, n =
10 pairs, *P < 0.05, Student’s t-test) or Rac1b C18Y (control: 0.24 ± 0.075 spines/μM, n = 7;
Rac1b C18Y: 0.19 ± 0.033 spines/μM, n = 13, P > 0.05, Student’s t-test) normalized and compared
to GFP expressing control neurons. Wilcoxon Rank Sum Test was used to compare across
independent conditions (i.e., Rac1b and Rac1b C18Y in E, *P < 0.05).

71
We expressed Rac1b, a constitutively active form of Rac1
183
, and Rac1b C18Y in CA1 pyramidal
neurons for 3 days. Consistent with its constitutively active feature, Rac1b expression led to a 4-
fold increase in AMPAR-eEPSC amplitude, nearly twice that seen with wild-type Rac1
(Figures 3.2C, 3.3A). Rac1b also resulted in a 2.5-fold increase in NMDAR-eEPSCs, an effect that
was not seen with wild-type Rac1 (Figures 3.2D, 3.3B). If C18Y prevents GTP from activating
Rac1b, expression of Rac1b C18Y should prevent the constitutive activation and cause Rac1b
C18Y and Rac1 C18Y to become functionally equivalent. We found that expression of Rac1b
C18Y phenocopied Rac1 C18Y, producing a ~60% reduction in AMPAR-eEPSC amplitude
compared to control neurons with no effect on NMDAR-eEPSC amplitude (Figures 3.3A-B). We
also found that Rac1b and Rac1b C18Y overexpression do not alter PPF, indicating that neither
manipulation modified presynaptic neurotransmitter release (Figure 3.3C). We then assessed the
cause of alteration in AMPAR-eEPSCs and NMDAR-eEPSCs following Rac1b and Rac1b C18Y
expression. CV analysis suggests that Rac1b, like Rac1, increases AMPAR function at all
functional gluatamatergic synapses (Figure 3.3D). Rac1b C18Y, like Rac1 C18Y, inhibits
AMPAR-eEPSC amplitude by reducing the number of glutamatergic synapses that contain
functional AMPARs (Figure 3.3D). CV analysis of increased NMDAR-eEPSC amplitude
following Rac1b expression results from an increase in quantal content, suggesting an increase in
the number of synapses that express NMDARs (Figure 3.3E). Consistent with this finding, changes
in NMDAR-eEPSCs commonly coincide with changes in glutamatergic synapse number. Because
of this, we examined dendritic spine density following Rac1b expression. We found that Rac1b
expression produces a nearly 2-fold increase in dendritic spine density. This increase accounts for
the bulk of the NMDAR phenotype. Rac1b C18Y on the other hand prevented the synaptogenic
effects of Rac1b (Figure 3.3E). Taken together, our data show that Rac1b C18Y prevents Rac1b’s
72

73
Figure 3.4
Rac1 C18Y expression prevents long-term potentiation (LTP) induction.
A-B, Plots of AMPAR-eEPSC amplitude of untransfected neurons (black) and neurons transfected
with Rac1 (green) or Rac1 C18Y (red) normalized to the mean AMPAR-eEPSC amplitude before
LTP induction (arrow). Sample AMPAR-eEPSC current traces from control (black) and
transfected (green for Rac1, red for Rac1 C18Y) neurons before and after LTP inductions are
shown (Scalebars: 20 ms, 20 pA). The bar graph shows individual LTP magnitude (mean ± SEM),
Wilcoxon Signed Rank Test. Wilcoxon Rank Sum Test was used to compare across independent
conditions (i.e., GFP and Rac1 C18Y in B, *P < 0.05). A, Wild-type Rac1 expression in CA1
pyramidal neurons does not significantly affect LTP induction (control: n = 7 neurons; Rac1: n =
6 neurons). B, Rac1 C18Y prevents LTP induction (control: n = 8 neurons; Rac1 C18Y: n = 6.

74
effects on synaptic transmission and largely phenocopies Rac1 C18Y. Thus, we conclude that
C18Y mutation prevents GTP from activating Rac1.
Rac1 C18Y prevents LTP induction
LTP is required for learning and memory formation in the brain. Rac1 activation has been
implicated in LTP, and we have recently shown that the Rac1 GEFs Kalirin and Trio are critical
for LTP induction
56
. LTP results in glutamatergic synapse unsilencing
178
, and here we find that
expression of a severe ID-related mutation in Rac1 results in an increased number of silent
synapses. Thus, we were interested in whether this ID-related mutation in Rac1 influences the
induction of LTP. To answer this question, in utero electroporation of embryonic day (E) 15 mice
was used to express either Rac1 or Rac1 C18Y in hippocampal CA1 pyramidal neurons during
early periods of brain development. Acute hippocampal slices were prepared from juvenile mice
(postnatal day (P) 21-29 mice). We then used a “pairing” LTP induction protocol (see “Materials
and Methods” section) that produces robust NMDAR-dependent LTP to examine LTP induction
in CA1 pyramidal neurons overexpressing Rac1 or expressing Rac1 C18Y. We found that
overexpression of wild-type Rac1 produced a modest but not statistically significant increase of
LTP compared to untransfected control neurons (Figure 3.4A). In contrast, we found that
expression of Rac1 C18Y abolished LTP induction (Figure 3.4B). Given the importance of LTP
in information storage in the brain, our results suggest that Rac1 C18Y’s ability to suppress LTP
induction likely contributes to the severe ID of this individual.
2.5 Discussion
Accumulating evidence points to glutamatergic synapse dysregulation and Rac1-mediated
actin polymerization as convergence points of a number of pathways implicated in ASD/ID
184
.
Rac1 is directly involved in glutamatergic synapse function and plasticity. However, the impact of
75
ASD/ID-related mutations in Rac1 on glutamatergic synapses has not been investigated. In this
study, we characterize the impact of a severe ID-related mutation in Rac1, C18Y, on glutamatergic
synapse function. Our modeling shows that Rac1 C18Y likely prevents GTP activation of Rac1.
Our electrophysiological data in hippocampal slices show that wild-type Rac1 overexpression
selectively increases AMPAR-mediated neurotransmission across all functional glutamatergic
synapses. This increase may be explained by either the insertion of additional AMPARs into
synapses or modification of existing synaptic AMPARs. In contrast, we found that Rac1 C18Y
expression reduces AMPAR-eEPSC amplitude compared to control neurons. The impact of Rac1
and Rac1 C18Y transfection on neurons did not differ across synaptic developmental time points
and these manipulations did not affect spine density or presynaptic release. CV analysis revealed
that reductions in AMPAR-mediated neurotransmission caused by Rac1 C18Y expression result
from a reduction in the number of glutamatergic synapses that contain functional AMPARs. We
also found that the C18Y mutation prevents the activity of a constitutively active Rac1, strongly
suggesting that the C18Y mutation prevents GTP activation of Rac1. This finding is consistent
with our mutational modeling. The precise mechanism behind Rac1 C18Y’s ability to increase the
number of AMPAR-lacking synapses in neurons is unclear. We believe this effect most likely
arises from either a dilution of endogenous wild-type synaptic Rac1 with a nonfunctioning form
of Rac1 or the ability of Rac1 C18Y to bind to Rac1-activating GEF proteins and reduce their
availability to bind to and activate functional Rac1 molecules. The individual harboring the Rac1
C18Y mutation is heterozygous for the mutation and, as a result, cells express both wild-type Rac1
and Rac1 C18Y. Thus, potential methods of competition between wild-type Rac1 and Rac1 C18Y
are relevant.
76
Rac1 has been implicated in the induction of LTP, the cellular basis for learning and
memory formation
185,186
. We have recently found that two Rac1-activating GEF proteins, Kalirin
and Trio, are required for CaMKII-dependent LTP induction
56
. Increases in Rac1 activity in
dendritic spines are thought to promote actin-mediated structural changes of synapses that underlie
synaptic AMPAR insertion during LTP
56
. We reasoned that this intellectual disability-related
C18Y mutation in Rac1 may prevent Kalirin and Trio-mediated upregulation of Rac1 activity
during LTP and thus inhibit LTP induction. Indeed, we find that in marked contrast to wild-type
Rac1 overexpression, neuronal Rac1 C18Y expression prevents the induction of LTP. It is
therefore likely that Rac1 C18Y’s potent ability to inhibit the induction of LTP contributes to the
ID observed in this individual.
Synaptic Rac1 dysregulation is now believed to contribute to ASD/ID-related behavioral
phenotypes in a number of established animal models of ASD/ID
184
. Shank3 knockout mice, for
example, are a well-established model for ASD/ID that display ASD/ID associated behaviors and
exhibit pronounced synaptic dysfunction. A recent study showed that enhancing Rac1 function
through the inhibition of a negative regulator of Rac1 activation alleviated both the synaptic and
ASD-related behavioral phenotypes of these mice
119
. Fragile X syndrome, the most common
inherited form of Autism, may also stem from synaptic Rac1 dysregulation. Fragile X syndrome
is caused by the reduced expression of FMRP, a protein translation repressor protein encoded by
the Fmr1 gene. Fmr1 knockout mice exhibit increased glutamatergic synapse density as well as
behaviors that are similar to the human condition
94
. These mice were found to have increased Rac1
expression
187
. It was recently shown that inhibiting the Rac1 effector protein, PAK, reversed both
the synaptic and behavioral phenotypes observed in Fmr1knockout mice
118
. Taken together, such
evidence suggests that both Rac1 hypo and hyperfunction are responsible for pathological
77
conditions at synapses that give rise to ASD/ID-related disorders. Consistent with this idea, we
have recently discovered an ASD/ID-related de novo mutation hotspot in the Rac1 activation
domain of Trio. Mutations in this domain either reduce or increase Trio’s ability to activate Rac1
122
.
Ultimately, dysregulation of synaptic Rac1 activity levels in either direction is likely to produce
disruption of important regulatory mechanisms at glutamatergic synapses. For example, Rac1 is
an important regulator of Cyfip1 function, with elevated Rac1 activation levels triggering a shift
from Cyfip1’s involvement in translational regulation toward a more direct role in synaptic actin
regulation
188
. In the present study we find that a Rac1 mutation in an individual with severe ID
results in a synaptic phenotype nearly identical to that observed with hypofunctional ASD-related
mutations in Trio. Thus, the Trio-Rac1 pathway may be a promising candidate for convergence of
a number of ASD/ID associated factors, given that pathological mutations in the Trio-Rac1 axis
of synaptic regulation produce a diverse array of glutamatergic synapse phenotypes that are similar
to the varied synaptic phenotypes observed in animal models of ASD/ID. It will be necessary to
determine the prevalence of altered Trio-Rac1 pathway function in patients with ASD/ID and
establish whether Trio/Rac1-related therapies can be applied to reverse ASD/ID-related behavioral
phenotypes in patients with these disorders.

78
Chapter 4: Autism Spectrum Disorder/Intellectual Disability-associated
Mutations in Trio Disrupt Neurolign1-mediated synaptogenesis
4.1 Abstract
We recently identified an Autism Spectrum Disorder/Intellectual Disability (ASD/
ID)-related de novo mutation hotspot in the Rac1 activating GEF1 domain of the protein Trio.
Trio is a Rho guanine nucleotide exchange factor (RhoGEF) that is essential for glutamatergic
synapse function. An ASD/ID-related mutation identified in Trio's GEF1 domain, Trio D1368V,
produces a pathological increase in glutamatergic synaptogenesis, suggesting that Trio is coupled
to synaptic regulatory mechanisms that govern glutamatergic synapse formation. However,
the molecular mechanisms by which Trio regulates glutamatergic synapses are largely
unexplored. Here, using biochemical methods we identify an interaction between Trio and
the synaptogenic protein Neuroligin 1 (NLGN1) in the brain. Molecular biological approaches
were then combined with super resolution dendritic spine imaging and whole-cell voltage clamp
electrophysiology in male and female rats to examine the impact ASD/ID-related Trio mutations
have on NLGN1-mediated synaptogenesis. We find that an ASD/ID-related mutation in Trio's
8th spectrin repeat region, Trio N1080I, inhibits Trio's interaction with NLGN1 and
prevents Trio D1368V-mediated synaptogenesis. Inhibiting Trio's interaction with NLGN1
via Trio N1080I blocked NLGN1-mediated synaptogenesis and increases in synaptic NMDA
receptor function but not NLGN1-mediated increases in synaptic AMPA receptor function.
Finally, we show that the aberrant synaptogenesis produced by Trio D1368V is dependent
on NLGN signaling. Our findings demonstrate that ASD/ID-related mutations in Trio are able
to pathologically increase as well as decrease NLGN-mediated effects on glutamatergic
neurotransmission, and point to a NLGN1-Trio interaction as part of a key pathway involved in
ASD/ID etiology.
79
4.2 Introduction
Increasing evidence suggests that Autism Spectrum Disorder (ASD) pathogenesis can be
attributed to excitatory synapse dysfunction
33,160,189
. Glutamatergic synapses, the primary
excitatory synapses in the brain, are formed on mushroom-like protrusions called dendritic spines.
These spines are filled with and supported by a dense skeletal meshwork of actin filaments. Exome
sequencing studies reveal that many ASD risk factors influence dendritic spine structure and
function
29,184,190
. The small GTPase Rac1, a key regulator of actin polymerization in dendritic
spines, has also been proposed as a promising candidate of convergence of many ASD risk
factors
117,119,172,191-193
.
The Rho guanine nucleotide exchange factor (RhoGEF) Trio and its paralog, Kalirin, play
an essential role in glutamatergic synapse structure and function through their ability to activate
Rac1
55,56,58,194,195
. We recently identified a cluster of disruptive ASD/ID-related de novo mutations
in the Rac1 activating domain of Trio, GEF1
122
. One missense mutation, Trio D1368V, increases
Trio’s ability to activate Rac1 and results in a pathological increase in glutamatergic
synaptogenesis. The molecular mechanisms by which Trio D1368V facilitates synaptogenesis is
unresolved. Understanding how ASD/ID-related mutations give rise to elevated synaptogenesis is
of great interest given that pathological elevations in glutamatergic synapse formation have been
observed in a number of animal models of ASD/ID as well as in many individuals with
ASD/ID
59,94,96,162
.
To identify ASD/ID-related Trio protein interactions that are involved in synaptogenesis,
we investigated the ASD/ID-related mutation Trio N1080I within Trio’s 8th spectrin repeat.
Spectrin repeats support protein interactions that govern a protein’s participation in cellular
regulatory pathways
196
. Here we found that the aberrant synaptogenesis produced by the Trio
80
D1368V mutation was abolished by adding the N1080I mutation to our Trio D1368V expression
construct. This finding led us to ask whether Trio N1080 supports Trio’s ability to interact with
synaptogenic proteins. We have shown previously that both Trio and Kalirin-7 bind to the highly
synaptogenic protein Neuroligin 1 (NLGN1) in the brain
46,85
. While Kalirin was found to bind to
NLGN1 more strongly than Trio, an interaction between Trio and NLGN1 was detectable
46
.
NLGN1 is a member of the Neuroligin (NLGN) protein family. NLGNs are well-established
postsynaptic cell-adhesion molecules that share considerable similarity in protein domain structure.
Through their interaction with presynaptic neurexins, NLGN proteins play essential and
overlapping roles in glutamatergic synaptogenesis
85
. In the present study we confirm that Trio
binds to NLGN1 in the brain and supports NLGN1 function. This relationship between Trio and
NLGN1 is of particular interest given that both Trio and NLGN1 are implicated in ASD/ID-related
disorders
197,198
. Here, we discover that Trio N1080I inhibits Trio’s ability to bind to NLGN1, and
that Trio N1080I expression inhibits NLGN1-mediated synaptogenesis and NLGN1’s influence
on synaptic NMDA receptor function but not synaptic AMPA receptor function. Together, these
findings led us to ask whether Trio D1368V’s ability to increase synaptogenesis is supported by
NLGN1. As predicted, we find that inhibition of NLGN signaling in neurons prevents Trio
D1368V from generating new synapses. Thus, elevated Rac1 activation produced by the D1368V
mutation serves to boost NLGN-mediated glutamatergic synapse formation. Altogether, our
findings demonstrate that that ASD/ID-related mutations in Trio are able to increase as well as
decrease NLGN-mediated synaptogenesis, and point to a NLGN1-Trio interaction as part of a key
pathway involved in ASD/ID-related disorders.
4.3 Materials and Methods
Electrophysiology
81
400 μm organotypic hippocampal slice cultures were prepared from P6-8 Sprague-Dawley
male and female rat pups as described previously
199
. Culture media was exchanged every other
day. Sparse biolistic transfections of organotypic slice cultures were carried out on DIV1 as
described
200
. Construct expression was confirmed by GFP and mCherry co-transfection. Paired
whole-cell recordings from transfected neurons and non-transfected control neurons were
performed on DIV7 slices. This study was carried out in accordance with the National Institutes
of Health (NIH) Guide for the Care and Use of Laboratory Animals and the protocol was approved
by the University of Southern California Institutional Animal Care and Use Committee.
Coefficient of Variation Analysis
Coefficient of variation (CV) analysis was performed on AMPAR-eEPSC by comparing
the change in eEPSC variance with the change in mean amplitude as previously
described
149,151,152,193,201
. Briefly, the mean (M) and standard deviation (SD) of eEPSC were
measured, normalized and plotted for a concurrent set of stimuli from a control and its neighboring
transfected cell. It has been shown 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 × presynaptic release probability, Pr; CV
−2
= nPr/(1 − Pr)
149,150,177
. CV analysis is
here 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. Simple linear regression were
obtained using the least square method. Regression line fall on or near the 45° (y = x) line suggest
changes in quantal content while regression line approaching the horizontal line (y = 1) suggest a
change in quantal size. Unsilencing of synapses can mimic an increase in the number of release
sites when presynaptic release probability is unchanged.
82
Spine density analysis
For spine density analysis, control and experimental CA1 pyramidal neurons in
organotypic hippocampal slice cultures made from P6-8 rat pups were biolistically transfected with
FUGW-GFP and pCAGGS-IRES-mCherry constructs approximately 18–20 h after plating.
Images were acquired at DIV7 using super-resolution microscopy (Elyra Microscope System,
Zeiss, Oberkochen, Germany). Z-stacks were made of 30 µm sections of secondary apical
dendrites ~30 µm from the soma. Images were acquired with a 100× oil objective (100×/1.46) in
SIM mode using a supplied 42 μm SIM grating and processed and reconstructed using supplied
software (Zen, Zeiss). An experimenter, blinded to the condition of the image, performed image
analysis on individual sections using ImageJ to count spines extending laterally from the dendrite.
Immunoblotting and immunoprecipitation
For co-immunoprecipitation experiments of endogenous protein, Sprague Dawley outbred
adult rat brains (>3 months of age) were homogenized and fractionated as described previously
46
.
Brains were homogenized in ice cold TEVP buffer containing 320 mM sucrose with protease and
phosphatase inhibitors. Samples were centrifuged and supernatants were centrifuged once more to
collect crude synaptosomes (P2). P2 pellets were solubilized for 30 mins at 37 C. The resultant
solution was incubated on ice for 30 mins and centrifuged again for 20 min. The supernatant was
used for immunoprecipitation of endogenous proteins. For immunoprecipitation of proteins
expressed in heterologous cells, HEK293T cells were maintained and processed as described
previously
46
. Briefly HEK293T cells were transfected using Lipofectamine 2000. 48hrs following
transfection cells were washed and lysed. Lysates were rocked for 1 h and centrifuged.
Supernatants were collected and used for immunoprecipitation with HA-beads incubated overnight
at 4°C. Blots were quantified using FIJI.
83
Rac1 and RhoA activity assays
Rac1 activity was assessed using the Thermo Scientific Active Rac1 Pull-Down and
Detection Kit (cat# 16118) according to manufacturer instructions. Briefly, HEK293T cells were
transfected with 5 ug of either WT trio-9 or N1080I trio-9. After 18-24 hours, HEK293T cells
were lysed and subjected to Rac1 pulldown and run on 12% SDS-PAGE, for Rac1 blotting, or 6%
SDS-PAGE, for Trio-9 blotting, at 60-100 V for 1.5 hours, then transferred in 10% methanol buffer
at 350 mA for 1.5 hours. Blots were probed with manufacturer provided Rac1 antibody or Trio
antibody
46
at 4°C overnight, then incubated with HRP-conjugated secondary antibody at room
temperature for 1 hour. Western blot images were collected on the BioRad ChemiDOC imaging
system. RhoA activity was assessed using the Cell Signaling Technology Active Rho Detection
kit (cat# 8820S) according to manufacturer instructions. Sample preparation and western blotting
was accomplished as in the Rac1 activity assay.
4.4 Results
The ASD/ID-related mutation Trio N1080I disrupts Trio protein function and blocks
synaptogenesis mediated by the ASD/ID-related mutation Trio D1368V
We recently discovered a large cluster of disruptive ASD/ID-related de novo mutations in
Trio’s Rac1 activating domain, GEF1 (Figure 4.1A)
122
. Outside of increased Rac1 activation, the
molecular mechanism supporting the synaptogenic properties of the hyperfunctional mutation Trio
D1368V remain unknown. This is due to our very limited current understanding of the
glutamatergic synapse regulatory pathways that involve Trio. Interestingly, ASD/ID-related de
novo mutations also reside outside of Trio’s GEF domains (Figure 4.1A). Such mutations likely
impact protein-protein interactions that govern Trio’s influence on glutamatergic synapses. One
84

85
Figure 4.1
The ASD/ID-related mutation Trio-9 N1080I disrupts Trio protein function and blocks
synaptogenesis mediated by the ASD/ID-related mutation Trio-9 D1368V.
A, ASD/ID-related de novo mutations in Trio. NDD, Neurodevelopmental disorder. †-
www.decipher.sanger.ac.uk. B, Electrophysiology recording setup. C, Summary of AMPAR and
NMDAR-eEPSC amplitudes (mean ± SEM) for each condition tested normalized to their
respective neighboring untransfected paired control neurons (black bar). Significance was
determined by Wilcoxon Signed Rank Test in each condition. Gray bars showing the effects of
wild-type Trio-9 overexpression on AMPAR and NMDAR-eEPSC amplitude represent previously
published data
56
and are repeated here for clarity. Trio-9 N1080I expression failed to increase
AMPAR-eEPSC amplitude (n=9, p=0.8203). Expression of wild-type Trio-9 (previously
published) or Trio-9 N1080I (n=16, p=0.2114) does not affect NMDAR-eEPSC amplitude. The
ASD/ID-related mutant Trio D1368V significantly increased both AMPAR- (n=8, *p=0.0078) and
NMDAR-eEPSC amplitude (n=8, *p=0.0078). A mutant form of Trio-9 harboring both N1080I
and D1368V failed to increase either AMPAR (n=8, p=0.25) or NMDAR-eEPSC amplitudes (n=6,
p=1). Wilcoxon rank-sum tests were used to compare across independent conditions (i.e. AMPAR-
eEPSCs: Wild-type Trio-9 vs Trio-9 D1368V, ∗p =0.0093; Trio-9 N1080I D1368V vs Trio-9
D1368V, *p=0.0003; NMDAR-eEPSCs: Trio-9 N1080I D1368V vs Trio-9 D1368V, *p=0.0426).
D, Scatterplots showing the data for the individual conditions summarized in panel C. Open circles
represent individual paired recordings, and filled circles represent the means ± SEM. The traces
show representative currents for each condition, with the transfected cell in color and the control
cell in black (vertical scale bars, 20 pA; horizontal scale bars, 20 ms for AMPA, 50ms for NMDA).
E, Representative dendritic spine images from neurons transfected with Trio-9 D1368V and
86
double mutant Trio-9 N1080I D1368V are shown with their corresponding untransfected control
dendrites (Scale bars: 2 μm). The boxplots show the 25
th
, 50
th
and 75
th
percentiles of each condition
with the means indicated by diamonds. Trio-9 D1368V expression resulted an in increase in spine
density. (Control, n=6; D1368V, n=7. *p=0.0029, Student’s t-test). Trio-9 N1080I D1368V
expression failed to produce an increase in spine density. (Control, n=6; Trio N1080I D1368V,
n=7. p=0.7452, Student’s t-test). F, Trio N1080I has no effect on Trio-9’s ability to activate Rac1
(n=3, p=0.727 Student’s t-test) or RhoA (n=3, p=0.842, Student’s t-test). n.s.=not significant.

87
ASD/ID-related de novo missense mutation, Trio N1080I, resides within Trio’s 8th spectrin repeat
(Figure 4.1A) and was identified in an individual with severe phenotypes including no verbal
communication, hand stereotypies and aggressive episodes
202
. We were, therefore, interested in
studying Trio N1080I in an effort to better understand the importance of this spectrin repeat in
synaptic function and to uncover the molecular mechanism(s) that are disrupted by this ASD/ID-
related mutation. We first generated the N1080I mutation in Trio-9, the most abundant Trio
isoform in the brain
126,203
. In order to determine the impact of Trio-9 N1080I on synaptic function,
we expressed Trio-9 N1080I in CA1 pyramidal neurons of organotypic rat hippocampal slice
cultures using biolistic transfection. 6 days after transfection we recorded AMPA receptor and
NMDA receptor-evoked excitatory postsynaptic currents (AMPAR and NMDAR-eEPSCs) from
Trio-9 N1080I transfected neurons and neighboring untransfected (wild-type) neurons
simultaneously during stimulation of Schaffer collaterals (Figure 4.1B). This approach permits a
pairwise, internally controlled comparison of the consequences of the genetic manipulation
46,56,201
.
We have shown previously that overexpression of Trio-9 produces a two-fold increase in AMPAR-
eEPSC amplitude (Figure 4.1C)
122
. In contrast, expression of Trio-9 N1080I failed to increase
the amplitude of AMPAR-eEPSCs (Figure 4.1C-D). NMDAR-eEPSC amplitude was not affected
by expression of either Trio-9 or Trio-9 N1080I (Figure 4.1C-D). These data demonstrate that in
neurons Trio N1080I inhibits Trio’s influence on glutamatergic synapse function.
We have previously shown that, in contrast to wild-type Trio-9, expression of the
hyperfunctional ASD/ID-related Trio mutant, Trio-9 D1368V, in CA1 pyramidal neurons
markedly increases dendritic spine density as well as both AMPAR and NMDAR-eEPSC
amplitude
122
. One possibility is that the increased Rac1 activation caused by the D1368V mutation
amplifies a synaptogenic regulatory pathway involving Trio. It stands to reason that Trio’s
88
involvement in such a synaptogenic regulatory pathway may be mediated by Trio’s association
with upstream synaptogenic proteins through its spectrin repeat region. Given the importance of
Trio’s 8th spectrin repeat in Trio function, its implication in ASD/ID, and potential to support
protein-protein interactions, we were interested in whether disruption of this domain by Trio
N1080I might prevent Trio’s interaction with an upstream synaptogenic protein and prevent Trio
D1368V’s ability to form new glutamatergic synapses. As shown previously, we find here that
expression of Trio-9 D1368V in CA1 pyramidal neurons for 6 days leads to a nearly 5-fold increase
in AMPAR-eEPSC amplitude (Figure 4.1C-D), a ~3.5-fold increase in NMDAR-eEPSC amplitude
(Figure 4.1C-D), and a significant increase in dendritic spine density (Figure 4.1E). We then
introduced the N1080I mutation into Trio-9 D1368V generating a mutant form of Trio-9 harboring
both the D1368V mutation and the N1080I mutation. Remarkably, we found that adding the
N1080I mutation to Trio-9 D1368V completely blocked Trio-9 D1368V’s ability to increase both
AMPAR and NMDAR-eEPSC amplitude (Figure 4.1C-D) and its ability to increase dendritic
spine density (Figure 4.1E). We also find that the Trio-9 N1080I mutation does not affect the
ability of Trio-9’s GEF1 domain to activate Rac1 or the ability of Trio-9’s GEF2 domain to activate
RhoA (Figure 4.1F). Together, such data suggest that Trio N1080I prevents a protein-protein
interaction that is required for Trio D1368V-mediated synaptogenesis.
Trio N1080I inhibits Trio’s interaction with NLGN1 and blocks NLGN1-mediated synaptogenesis.
We recently found that Trio and its paralog Kalirin bind to NLGN1, a prominent driver of
glutamatergic synapse formation
46,204-206
. To confirm Trio’s interaction with NLGN1 in the brain,
we performed an immunoprecipitation assay of endogenous Trio from the P2 fraction of whole rat
brain homogenates. Indeed, our Co-IP assay revealed that Trio interacts with NLGN1 at synapses

89

90
Figure 4.2
Trio-9 N1080I inhibits Trio’s interaction with NLGN1 and blocks NLGN1 mediated
synaptogenesis.
A, Immunoblot analysis showing co-immunoprecipitation (Co-IP) of NLGN1 but not
synaptophysin (SYP) with Trio in adult rat P2 brain fractions. B, Left: Immunoblot analysis
showing Co-IP of HA-NLGN1 with Trio-9 or Trio-9 N1080I in HEK293T cells. Right: Total Trio-
9 and Trio-9 N1080I lysate levels (means ± SEM) normalized to control. Compared to wild-type
Trio, Trio-9 N1080I has significantly less interaction with NLGN1. *p=0.02 Student’s t-test. C-E,
Representative dendritic spine images from transfected neurons of each condition are shown with
their corresponding control image (Scale bars: 2 μm). Boxplots show the 25
th
, 50
th
and 75
th

percentiles of each condition with the means indicated by diamonds. C, Expression of NLGN1
resulted in an increase in dendritic spine density. (control, n=9; NLGN1, n=8. *p=0.0192,
Student’s t-test). D, Replacing endogenous Kalirin and Trio with wild-type Trio-9 supports
NLGN1 mediated enhancement in dendritic spine density. (control, n=6; Kalirin/Trio KD & Trio-
9 & NLGN1, n=5. *p=0.0141, Student’s t-test). E, Replacing endogenous Kalirin and Trio with
Trio-9 N1080I prevented NLGN1 from increasing dendritic spine density. (control, n=7;
Kalirin/Trio KD & Trio-9 N1080I & NLGN1, n=7. p=0.9691, Student’s t-test). KD: knockdown.
n.s.=not significant.
91
in vivo (Figure 4.2A). The absence of Trio binding to another synaptic protein, synaptophysin
(SYP), in this assay revealed that Trio’s interaction with NLGN1 was specific (Figure 4.2A).
Immunoprecipitation of Trio-9 with NLGN1 in HEK293 cells strongly suggests that a direct
interaction exists between these two proteins (Figure 4.2B). We then reasoned that Trio-9
N1080I‘s ability to prevent Trio-9 D1368V-mediated glutamatergic synaptogenesis may stem
from Trio N1080I inhibiting an interaction between Trio and NLGN1. To test this hypothesis, we
co-expressed Trio-9 N1080I and NLGN1 in HEK293 cells. Remarkably, we found that NLGN1’s
ability to co-immunoprecipitate Trio-9 N1080I was significantly reduced by ~50% when
compared to wild-type Trio (Figure 4.2B). Thus, Trio N1080I inhibits Trio’s ability to associate
with NLGN1.
We next examined whether NLGN1-mediated synaptogenesis is affected by Trio N1080I.
NLGN1 is essential in the formation of glutamatergic synapses and expression of NLGN1 in
neurons results in a significant increase in dendritic spine density
207
. We expressed NLGN1 for 6
days in CA1 pyramidal neurons of the hippocampus and found that this resulted in a ~40% increase
in spine density when compared to control neurons transfected with GFP (Figure 4.2C). In the
brain, both Trio and Kalirin associate with NLGN1 and thus both proteins may support NLGN1’s
effects on glutamatergic synapse formation. However, Trio is generally expressed in greater
abundance than Kalirin in neurons early in postnatal development and stands to play a larger role
in supporting NLGN1 effects on glutamatergic synapse formation during this time
123,125
. In order
to isolate the role Trio plays in NLGN1-mediated synaptogenesis and determine whether Trio can
support NLGN1-mediated synaptogenesis in the absence of Kalirin, we molecularly replaced
endogenous Kalirin and Trio with recombinant Trio-9. We transfected CA1 pyramidal neurons
with our previously validated RNAi’s against Kalirin and Trio
56
, a RNAi-resistant Trio-9 and our
92
NLGN1 expression construct. In these neurons, we observed an increase in dendritic spine density
that was nearly identical to NLGN1 overexpression alone in wild-type neurons (Figure 4.2D).
These results were consistent with Trio-9 supporting NLGN1’s ability to create new glutamatergic
synapses. In marked contrast, we found that replacing endogenous Trio and Kalirin with Trio-9
N1080I completely abolished NLGN1’s ability to increase dendritic spine density (Figure 4.2E).
Together, these data suggest that Trio is sufficient to support NLGN1-mediated synaptogenesis
and that inhibiting Trio’s ability to associate with NLGN1 prevents NLGN1’s ability to promote
new glutamatergic synapse formation.
Trio N1080I prevents NLGN1 from increasing NMDAR- but not AMPAR-mediated synaptic
transmission.
Our observation that Trio-9 N1080I expression prevents NLGN1-mediated increases in
dendritic spine number prompted us to perform a detailed electrophysiological examination of how
Trio N1080I affects NLGN1’s influence on glutamatergic synapse function. In addition to
increasing dendritic spine number, synaptogenesis produced by NLGN1 expression is manifested
as augmentations of synaptic function
204
. For example, we as well as others find that expression
of NLGN1 in CA1 pyramidal neurons results in a ~2.5 fold increase in AMPAR and NMDAR-
eEPSC amplitude (Figure 4.3A-B)
46,204
. First, to test whether Trio and Kalirin are necessary for
NLGN1-mediated increases in AMPAR and NMDAR-eEPSC amplitude we knocked down both
Kalirin and Trio in CA1 pyramidal neurons. As previously published
56
we found here that
simultaneous knockdown of Kalirin and Trio results in substantial reductions in AMPAR and
NMDAR-eEPSC amplitude (Figure 4.3A-C). We then tested the effect of NLGN1 expression on
this Kalirin/Trio double knockdown background. We found that simultaneous knockdown of
Kalirin and Trio prevented NLGN1-mediated increases in AMPAR- and NMDAR-eEPSC
93

94
Figure 4.3
Trio-9 N1080I prevents NLGN1 from increasing NMDAR but not AMPAR-mediated
synaptic transmission.
Summary of AMPAR- A, and NMDAR-eEPSC B, amplitudes (mean ± SEM) for each condition
tested normalized to their neighboring untransfected paired control neurons (black bar). Bar
showing the NLGN1 expression phenotype
46
was previously published and is repeated here for
clarity. Significance was determined by Wilcoxon Signed Rank Test in each condition. Aligned
control graphs show fold eEPSC amplitude change of NLGN1 expression on the wild-type
background vs. the Trio-9 N1080I replacement background. C-H, Scatterplots showing the
individual conditions summarized in panels A and B. Open circles represent individual paired
recordings, and filled circles represent the means ± SEM. The traces show representative currents
for each condition, with the transfected cell in color and the control cell in black (vertical scale
bars, 20 pA; horizontal scale bars, 20 ms for AMPA, 50ms for NMDA). C, Knocking down Kalirin
and Trio significantly reduced AMPAR and NMDAR-eEPSC amplitudes (AMPAR-eEPSC, n=8,
*p=0.0078; NMDAR-eEPSC, n=7, *p=0.0312) D, NLGN1 expression on the Kalirin and Trio
double knockdown did not increase either AMPAR-eEPSC or NMDAR-eEPSC amplitude
compared to Kalirin and Trio double knockdown. (Kalirin/Trio KD & NLGN1: AMPAR-eEPSC,
n=8, *p=0.0078; NMDAR-eEPSC, n=7, p=0.2188; Kalirin/Trio KD versus Kalirin/Trio KD &
NLGN1 (Wilcoxon Rank Sum Tests): AMPA-eEPSC, p=1, NMDAR-eEPSC, p=0.9015) E,
Replacing Kalirin and Trio with wild-type Trio-9 produced AMPAR- and NMDAR-eEPSC
amplitudes that were similar to paired controls (AMPAR-eEPSCs, n=8, p=0.6406; NMDAR-
eEPSCs, n=6, p=0.2807). F, When Kalirin and Trio were replaced by wild type Trio-9, expression
of NLGN1 increased AMPAR and NMDAR-eEPSC amplitudes compared to wild-type controls
95
(AMPAR-eEPSCs, n=8, *p=0.0019; NMDAR-eEPSCs n=7, *p=0.0039) and neurons where
Kalirin and Trio were replaced with Trio-9 (AMPAR-eEPSCs, *p=0.0062; NMDAR-eEPSCs
*p=0.0119, Wilcoxon Rank Sum Tests). G, Replacing Kalirin and Trio with Trio-9 N1080I
produced AMPAR- and NMDAR-eEPSC amplitudes that were similar to paired controls
(AMPAR-eEPSCs, n=11, p=0.5195; NMDAR-eEPSCs, n=8, p=0.9453). H, NLGN1 expression
on the Trio-9 N1080I replacement background did not increase AMPAR-eEPSC amplitude
compared to paired control neurons (n=9, p=0.2031, Wilcoxon Rank Sum Tests) but produced a
significant increase relative to replacing Kalirin and Trio with Trio-9 N1080I (*p=0.0465,
Wilcoxon Rank Sum Tests A). NLGN1 expression on the Trio-9 N1080I replacement background
did not affect NMDAR-eEPSC amplitude compared to paired control neurons (n=8, p=0.3125,
Wilcoxon Rank Sum Tests) or relative to replacing Kalirin and Trio with Trio-9 N1080I (p=0.8785,
Wilcoxon Rank Sum Tests B). KD: knockdown. I, Coefficient of Variation (CV) analysis of
AMPAR-eEPSCs from pairs of control neurons and neurons where NLGN1 was expressed on the
Trio-9 N1080I molecular replacement background. CV-2 ratios are graphed against the mean
amplitude ratio for each pair. Filled circles showed mean  SEM. Dashed lines show linear
regression and highlighted region represents 95% confidence interval. (Kalirin/Trio KD & Trio
N1080I & NLGN1, n=9 pairs, R2=0.787, *p=0.0014, Simple Linear Regression). n.s.=not
significant.

96
amplitude (Figure 4.3A,B,D). These data demonstrate that Kalirin and Trio are required for
NLGN1-mediated augmentation of AMPAR and NMDAR-mediated synaptic transmission.
Given that Kalirin and Trio are paralogous proteins that both interact with NLGN1, we
reasoned that Kalirin and Trio should be capable of supporting NLGN1-mediated synaptogenesis
independently. We have shown previously that Kalirin alone can support NLGN1-mediated
increases in AMPAR- and NMDAR-eEPSC amplitude in the absence of Trio
46
. Here, we were
interested in whether Trio is able to support NLGN1-mediated increases in AMPAR- and
NMDAR-eEPSC amplitude in the absence of Kalirin. To answer this question we first expressed
recombinant Trio-9 on a Trio and Kalirin double knockdown background and found that molecular
replacement of Kalirin and Trio with recombinant Trio-9 restored normal glutamatergic
transmission (Figure 4.3A,B,E). This result is consistent with our previously published results
56

and illustrates that Trio-9 is able to substitute for Kalirin in supporting basal synaptic transmission.
We then examined the effect of NLGN1 expression on this Trio-9 replacement background to
determine whether NLGN1 can augment glutamatergic synapse function through Trio in the
absence of Kalirin. We found that NLGN1 expression on this Trio-9 replacement background
produced robust increases in both AMPAR- and NMDAR-eEPSC amplitude when compared to
both paired untransfected wild-type neurons and neurons where Kalirin and Trio were molecularly
replaced with Trio-9 (Figure 4.3A,B,F). These results were consistent with the effects we observed
with this genetic manipulation on dendritic spine density (Figure 4.2D) and demonstrate that Trio
is able to support NLGN1-mediated synaptogenesis in the absence of Kalirin.
We were then interested in whether Trio N1080I affects NLGN1’s ability to augment
glutamatergic neurotransmission. To answer this question, we first co-transfected CA1 pyramidal
neurons with our Kalirin and Trio RNAi and our Trio-9 N1080I expression construct. We found
97
that molecularly replacing Kalirin and Trio with Trio-9 N1080I resulted in AMPAR-and NMDAR-
eEPSCs that were similar to untransfected neurons (Figure 4.3A,B,G). In contrast to molecular
replacement with wild-type Trio, we found that molecular replacement with Trio-9 N1080I
completely blocked NLGN1’s ability to increase NMDAR-eEPSC amplitude (Figure 4.3B,H).
This result was also consistent with the effects we observed with this genetic manipulation on
dendritic spine density (Figure 4.2E), given that NMDAR-eEPSC amplitude phenotypes are often
coupled with changes in spine density
56,122,193,208
. However, molecular replacement with Trio-9
N1080I failed to prevent NLGN1’s ability to increase AMPAR-eEPSC amplitude. NLGN1
expression on the Trio-9 N1080I replacement background led to a greater than 2-fold increase in
AMPAR-eEPSC amplitude when compared to AMPAR-eEPSC of Trio-9 N1080I replacement
neurons that were not transfected with NLGN1 (Figure 4.3A,H). The magnitude of this increase
in AMPAR-eEPSC amplitude was nearly identical to that observed when NLGN1 is expressed on
a wild-type background (Figure 4.3A). Given that this increase in AMPAR-eEPSC amplitude
occurred in the absence of increased spine number and NMDAR-eEPSC amplitude we conclude
that AMPAR function is augmented at existing synapses. This increase in AMPAR-eEPSC
amplitudes could be due to a reduction in the number of AMPAR-lacking (“silent”) glutamatergic
synapses, or alternatively due to a uniform modification in AMPAR-mediated synapse
transmission across all functional synapses. To determine the source of the increase in AMPAR-
eEPSC amplitude when expressing NLGN1 on Trio N1080I replacement background, we
performed coefficient of variation analysis (CV analysis) on the AMPAR-eEPSC amplitudes of
this condition. Coefficient of variation analysis can be used to determine the quantal parameters
of glutamatergic transmission in control and transfected neurons. By comparing the normalized
variance in AMPAR-eEPSC amplitudes from two neurons receiving the same stimulus, it is
98
possible to estimate relative quantal size and quantal content
149,150,152
. Changes in quantal size
precisely change both the mean eEPSC and the variance such that the normalized ratio of
mean2/variance, also known as coefficient of variation (or CV
−2
), remains constant. Changes in
quantal size cause the marker of the mean to fall on the horizontal line seen in Figure 4.3I and, in
the context of this preparation, indicate a change in the number of glutamate receptors at all
synapses. In contrast, changes in quantal content will produce proportional changes of equal
magnitude in CV
−2
and mean eEPSC amplitudes that cause the marker of the mean to fall close to
the diagonal line. Here, changes in quantal content indicate a change in the number of synapses
expressing glutamatergic receptors. We observed proportional increases in CV
−2
and mean
AMPAR-eEPSC amplitude following NLGN1 expression on the Trio N1080I molecular
replacement background (Figure 4.3I). This result identified a clear increase in quantal content
rather than quantal size as responsible for the increase in AMPAR-eEPSC amplitude we observe.
Therefore, we conclude that NLGN1 expression on the Trio N1080I replacement background
results in an increase in the number of synapses that contain AMPARs. Altogether, our results
show that the ASD/ID-related mutation Trio N1080I leads to a selective inhibition of NLGN1’s
ability to create new synapses while preserving the ability of NLGN1 to convert AMPAR lacking
silent synapses into AMPAR-containing functional synapses.
NLGN signaling is required for Trio D1368V-mediated synaptogenesis.
Above we show that the ASD/ID-related mutation Trio N1080I inhibits NLGN1’s
association with Trio-9. We also show that Trio-9 N1080I prevents the synaptogenesis caused by
the hyperfunctional ASD/ID-related mutation Trio-9 D1368V. Together, these data are consistent
with the idea that Trio D1368V pathologically increases glutamatergic synapse formation by
amplifying NLGN1-mediated synaptogenesis. If NLGN1 signaling is in fact required for the
99

100
Figure 4.4
Figure 4. NLGN signaling is required for Trio-9 D1368V-mediated synaptogenesis.
Summary of AMPAR A and NMDAR-eEPSC B amplitudes (mean ± SEM) for each condition
tested normalized to their neighboring untransfected paired control neurons (black bar). Bars for
the Trio-9 D1368V expression phenotype were shown in Figure 1 and are repeated here for clarity.
Significance was determined by Wilcoxon Signed Rank Test in each condition. (AMPAR-eEPSCs:
NLGN-miRs, n=6, *p=0.0313; NLGN-miRs & Trio-9 D1368V, n=9, p=0.0977; NLGN miRs &
NLGN1-ΔC, n=7, *p =0.0390; NLGN-miRs & NLGN1-ΔC & Trio-9 D1368V, n=9, p=0.2031.
NMDAR-eEPSCs: NLGN-miRs, n=6, *p=0.0355; NLGN-miRs & Trio-9 D1368V, n=7, p=0.2969;
NLGN-miRs & NLGN1-ΔC, n=6, p=0.2188; NLGN-miRs & NLGN1-ΔC & Trio-9 D1368V, n=8,
p=0.1094.) Expression of Trio-9 D1368V did not increase AMPAR or NMDAR-eEPSC amplitude
on the NLGN-miRs background (AMPAR-eEPSC, p=1; NMDAR-eEPSC, p=0.366, Wilcoxon
Rank Sum Tests) or on the NLGN1-ΔC replacement background (AMPAR-eEPSC, p=0.9626;
NMDAR-eEPSC, p=0.9551, Wilcoxon Rank Sum Tests). C-F, Scatterplots showing the
individual conditions summarized in panels A and B. Open circles represent individual paired
recordings, and filled circles represent the means ± SEM. The traces show representative currents
for each condition, with the transfected cell in color and the control cell in black (vertical scale
bars, 20 pA; horizontal scale bars, 20 ms for AMPA, 50ms for NMDA). KD: knockdown. n.s.=not
significant. G, Model illustrations: NLGN1 promotes synaptogenesis through its interaction with
Trio; Trio N1080I inhibits Trio’s interaction with NLGN1 and prevents NLGN1-mediated
synaptogenesis; Trio D1368V augments NLGN1-mediated synaptogenesis through increased
Rac1-mediated actin polymerization.

101
pathological synapse formation produced by Trio D1368V, knocking down NLGN1 should
prevent Trio D1368V from enhancing glutamatergic synapse function. Due to overlapping
function, the presence of other NLGN isoforms is believed to compensate for the elimination of
individual NLGN isoforms at synapses
45,205
. Thus, elimination of the three major Neuroligin
proteins (NLGN1, 2, and 3) that influence synaptic formation using a chained NLGN1-3
microRNA construct (NLGN-miRs) has been used to study the role of NLGN signaling in
neurons
45,204,205
. It has been shown previously that knockdown of NLGN1-3 in CA1 pyramidal
neurons results in a reduction in glutamatergic neurotransmission
205
. Consistent with this study,
we find that expressing the chained NLGN-miRs construct in CA1 pyramidal neurons reduced
both AMPAR- and NMDAR-eEPSC amplitude (Figure 4.4A-C). We then expressed Trio-9
D1368V on this NLGN knockdown background and asked if expression of Trio-9 D1368V
enhances glutamatergic synapse transmission in the absence of NLGN protein function. We
observed a similar reduction in both AMPAR- and NMDAR-eEPSC amplitude compared to
NLGN1-3 knockdown (Figure 4.4A,B,D). These data demonstrate that NLGN signaling is
required for Trio-9 D1368V to induce the generation of new synapses.
As cell adhesion molecules, NLGN proteins are composed of an extracellular domain, a
transmembrane domain and intracellular cytoplasmic terminal (C-tail) domain, with the
extracellular domain being critical NLGN protein interactions with presynaptic Neurexin proteins.
In order to investigate whether NLGN1 intracellular signaling is specifically required for the
synapse formation produced by Trio-9 D1368V, we expressed a form of NLGN1 lacking its
intracellular C-tail domain (NLGN1-ΔC) together with the NLGN-miRs. This molecular
replacement of endogenous NLGN1-3 proteins with NLGN C-tail mutations was shown
previously to be necessary for isolating and studying the effects of NLGN C-tail truncation
102
mutations on synaptic function
205
. We find that NLGN1-ΔC molecular replacement resulted in
reduced glutamatergic synaptic transmission that was similar to the NLGN miRs alone (Figure
4.4A,B,E), demonstrating an elimination of NLGN1 signaling in these neurons. We then expressed
Trio-9 D1368V on this NLGN1-ΔC molecular replacement background and asked if expression
of Trio-9 D1368V could still enhance AMPAR and NMDAR-eEPSC amplitude. Similar to
expression of D1368V on NLGN knockdown background, we observed no significant difference
between NLGN1-ΔC molecular replacement and expression of Trio D1368V on the NLGN1-ΔC
molecular replacement background (Figure 4.4A,B,F). Altogether, our results demonstrate that
NLGN intracellular signaling is required for Trio D1368V-mediated synaptogenesis.
4.5 Discussion
Disruption of glutamatergic neurotransmission is widely believed to underlie the
development of ASD/ID-related disorders
33,121,160
. Trio resides within dendritic spines where it
promotes actin polymerization through its ability to activate Rac1
46,56,122
. This function is critical
for the formation and maintenance of glutamatergic synapses in the brain. The majority of
ASD/ID-related mutations identified in Trio are clustered in Trio’s GEF1 domain and alter Trio’s
ability to activate Trio’s downstream effector molecule Rac1. The ASD/ID-related mutation Trio
D1368V increases Trio’s ability to activate Rac1 and produces a potentially pathological increase
in glutamatergic synapse formation. These data suggest that Trio is coupled to molecular
mechanisms that govern glutamatergic synapse formation. However, prior to this study, the
molecular mechanisms responsible for Trio D1368V-mediated synaptogenesis have not been
explored. The spectrin repeat region of Trio likely supports protein-protein interactions that govern
Trio’s involvement in specific neuronal regulatory processes. We reasoned that this region may
support an interaction between Trio and proteins that promote glutamatergic synapse formation.
103
Another ASD/ID-related Trio mutation, Trio N1080I, is located in Trio’s 8th spectrin repeat and
inhibits Trio function without affecting Trio’s ability to activate small GTPases. Remarkably, we
found that a mutant form of Trio harboring both the N1080I and D1368V mutations does not
increase glutamatergic synapse formation. These data are consistent with the N1080I mutation
preventing Trio’s association with proteins involved in glutamatergic synaptogenesis, and thus
precluding Trio D1368V-mediated elevations in Rac1 signaling from making more synapses.
Trio and its paralog Kalirin interact with NLGN1, a postsynaptic protein that promotes
glutamatergic synaptogenesis through its interaction with presynaptic neurexin proteins. NLGN1
overexpression in neurons increases dendritic spine number and produces large increases in
AMPAR- and NMDAR-mediated synaptic currents. We have shown previously that Kalirin can
support NLGN1 function in the absence of Trio
46
. Here, we now show that Trio can support
NLGN1 function in the absence of Kalirin (Figure 4.4G). These data provide additional evidence
that Trio and Kalirin play overlapping roles in the regulation of postsynaptic function. However,
Trio is generally expressed at higher levels than Kalirin early in postnatal development with
Kalirin expression peaking later in development
123-125
. Thus, the influence Trio and Kalirin have
on synaptic regulation may vary as the brain develops. It is currently unknown why the brain
utilizes Trio over Kalirin at early developmental time points. Going forward it will be important
to identify the specialized roles these proteins play in synaptic regulation.
In the present study, we find that the Trio N1080I inhibits Trio’s ability to interact with
NLGN1. In contrast to wild-type Trio, we find that molecular replacement of Kalirin and Trio with
Trio N1080I prevented NLGN1 expression from increasing dendritic spine number and synaptic
NMDAR-mediated current amplitude. Thus, Trio N1080I prevents NLGN1 from creating new
synapses. Surprisingly, Trio N1080I molecular replacement did not prevent NLGN1’s ability to
104
increase synaptic AMPAR-mediated current amplitude. Such data suggest that Trio N1080I may
contribute to the development of ASD/ID by uncoupling NLGN1’s effects on glutamatergic
synapses, with this mutation selectively preventing NLGN1-mediated synaptogenesis but leaving
NLGN1’s ability to increase synaptic AMPAR function at existing synapses intact (Figure 4.4G).
Given that Trio N1080I does not completely prevent Trio’s ability to interact with NLGN1, we
believe that NLGN1’s ability to increase synaptic AMPAR function is supported by Trio N1080I.
Stronger interactions between Trio and NLGN1 may be required for NLGN1-mediated
synaptogenesis given the larger cytoplasmic volume of the dendrites relative to existing synapses.
The smaller cytoplasmic volume of existing synapses may result in higher Trio N1080I
concentrations relative to dendrites and allow functional NLGN1-Trio N1080I interactions to
occur within these structures. However, it is alternatively possible that NLGN1 increases synaptic
AMPAR function at existing synapses via a Trio-independent mechanism. We believe this
explanation is less likely given that molecularly replacing Kalirin and Trio with a mutant form of
Kalirin that completely eliminates its interaction with NLGN1 blocked NLGN1-mediated
increases in both AMPAR and NMDAR function
46
.
The ASD/ID-related mutation Trio-9 D1368V increases the ability of Trio-9 to activate
Rac1 and results in aberrant synaptogenic qualities not observed with wild type Trio-9. In the
present study, we show that Trio-9 D1368V-mediated synaptogenesis is blocked by inhibiting
Trio’s interaction with NLGN1 via N1080I. We also show that Trio-9 D1368V-mediated
synaptogenesis is blocked by eliminating intracellular NLGN signaling in neurons. Therefore, we
conclude that the synaptogenesis produced by Trio-9 D1368V is mediated through a pathological
increase in NLGN1-mediated glutamatergic synapse formation (Figure 4.4G). While Trio
D1368V-mediated synaptogenesis relies on NLGN signaling, it is interesting that molecular
105
replacement of Kalirin and Trio with Trio-9 N1080I largely restores baseline glutamatergic
neurotransmission. This finding is consistent with our previous study in which a form of Kalirin
that is unable to bind to NLGN1 is able to support largely normal baseline synaptic transmission
46
.
Such data may be explained by Kalirin and Trio interacting with other NLGN isoforms or other
postsynaptic trans-synaptic adhesion complex proteins which compensate for reduced Kalirin and
Trio binding to NLGN1. Given that deficits in glutamatergic neurotransmission are only observed
when NLGN1 expression is reduced during late embryonic development
45
, it is possible that
deficits in baseline synaptic transmission resulting from the Trio N1080I mutation only arise
transiently during periods of robust NLGN1-specific synaptogenesis during the earliest phases of
synaptic development. Going forward, it will be important to test this hypothesis in animal models
harboring the Trio N1080I mutation.
This work now provides examples of ASD/ID-related mutations in Trio that are able to
augment as well as inhibit NLGN-mediated signaling in neurons. Thus, either strengthening or
weakening this newly identified synaptic regulatory pathway stands to produce alterations in
glutamate-mediated synaptic communication between neurons that contribute to the development
of ASD/ID-related disorders. As a result, the development of therapeutics targeting NLGN1/Trio-
mediated glutamatergic synapse regulation may be a useful strategy in treating ASD/ID. In
addition to NLGN1, our proteomic work has revealed interactions between Trio and other ASD-
related genes as well (e.g. CTTNBP2, CRMP1 and DPYSL2)
46
. Future investigation of the
interactions between Trio and such ASD-linked proteins will be essential to our understanding of
how Trio dysfunction contributes to the development ASD/ID-related disorders.
106
Chapter 5: Conclusion
There is a strong genetic basis for ASD, but the risk architecture is very heterogeneous.
Understanding the cellular mechanisms of ASD-related mutations will help simplify the genetic
landscape of ASD-risk genes and thus, aid in the development of new strategies to treat patients
with a diverse array of ASD-causing factors. Increasing evidence suggests that ASD pathogenesis
can be attributed to dysregulation of glutamatergic synapse function
33,160,189
. The small GTPase
Rac1, a key regulator of glutamatergic synapse structure, has also been proposed as a promising
candidate of convergence of many ASD risk factors
117-119,172,192
. Here in this study, through
investigation of ASD-related de novo mutations, we have identified a Rho-GEF signaling pathway,
which is essential in the activation of GTPase Rac1, as a potential cellular mechanism contributing
to the development of ASD.
We have first queried databases of de novo mutations in patients in ASD-related disorders
and have identified a large number of mutations in protein Trio. Trio plays an essential role in
glutamatergic synapse structure and function by delicately regulating Rac1 activity via its
enzymatic unit GEF1 domain. The large number of ASD-related mutations we find clustered in
the GEF1 domain results in a very high level of genome-wide statistical confidence that these
mutations contribute to the development of ASD. We have then investigated the effect of the
clustered mutations on Rac1 activation and glutamatergic synapse transmission using a
combination of molecular biological approaches, Fluorescent lifetime imaging (FLIM),
computational modeling, super resolution dendritic spine imaging and whole-cell voltage clamp
electrophysiology.
We revealed that the majority of these mutations eliminated Trio’s ability to activate Rac1
and weakened synapse transmission. One mutation, on the contrary, distinctively increased Rac1
107
activation level and pathologically enhanced glutamatergic synaptogenesis. Based on the
observation that these mutations clustered in essential enzymatic unit in Trio, produce either hypo
functional or hyper functional forms of Trio and pathologically increases or decreases
glutamatergic synapse transmission, we reason that Trio-Rac1 pathway at glutamatergic synapses
is a possible key point of convergence of many ASD-related genes.
Despite mounting evidence of altered Rac1 function caused by ASD risk genes in
numerous ASD/ID models
117-119,122,172
, the influence of ASD-related mutation in Rac1 itself has
not yet been explored. In 2016, a meta-analysis of de novo mutations identified Rac1 as 1 of the
10 new Intellectual Disability (ID) related genes and reported a de novo Rac1 missense mutation
C18Y in an individual with severe ID
176
. ID is a neurodevelopmental disorder that often comorbid
with ASD. We have then examined the effect of the de novo mutation Rac1 C18Y on synapse
transmission and plasticity.
We discovered that the de novo mutation Rac1 C18Y results in selective reduction in
synaptic AMPA receptor function by occluding GTP-mediated activation of Rac1. We have further
uncovered that the severe ID-related mutation Rac1 C18Y prevents induction of long term
potentiation (LTP), the cellular mechanism underlying learning and memory formation. The
disruptive effect of Rac1 mutation C18Y directly on ID-related cellular mechanisms strongly
suggests that the mutation contributes to the development of ID symptoms.
The characterization of the de novo mutation in Rac1 itself further validated Rac1 signaling
and Trio-Rac1 pathway as a contributing factor to ASD-related disorders. However, the synaptic
upstream regulation of Trio-Rac1 pathway remains uncovered. The hyperfunction mutation
D1368V in the GEF1 domain of Trio results in enhanced synaptogenesis, suggesting that Trio is
108
coupled to synaptic regulatory mechanisms that govern glutamatergic synapse formation. We have
then investigated Trio’s interaction with synaptogenic proteins.
Using biochemical methods we identified an interaction between Trio and the synaptogenic
protein Neuroligin 1 (NLGN1) in the brain. We found that an ASD/ID related mutation in Trio’s
8
th
spectrin repeat region, Trio N1080I, inhibit Trio’s interaction with NLGN1 and blocked
NLGN1-mediated synaptogenesis. Remarkably, N1080I also prevented mutant Trio D1368V from
generating more synapses. We suspected that N1080I prevented Trio D1368V mediated
synaptogenesis by blocking the interaction between Trio and NLGN1. We have then examined
whether NLGN signaling is required for D1368V mediated synaptogenesis and found that the
aberrant synaptogenesis produced by Trio D1368V is dependent on NLGN signaling.
Together, our comprehension of the effect of ASD-related mutations on glutamatergic
synapse signaling points to disrupted Trio signaling as a potential core synaptic regulatory pathway
that is disrupted in ASD. Surprisingly we found no ASD-associated mutation in Trio’s nearly
identical paralog Kalirin despite that Trio and Kalirin are both RhoGEF proteins, share 90%
sequence identity and play similar role in glutamatergic synapse function
56
. Trio is highly
expressed in early postnatal development and decreases with age
123
while Kalirin does not peak
until adolescence
209
. Remarkably, Kalirin function has been implicated in later onset
neuropsychiatric and neurodegenerative disorders like schizophrenia and Alzheimer’s
diseases
158,210,211
. It appears that the expression profile of Trio and Kalirin match the age of onset
of disease in which they are involved in and disruption of RhoGEF signaling at different time
points of brain development contribute to distinct brain disorders.
Our study point to the importance of RhoGEF signaling in glutamatergic synapse function
and in variety of brain-related disorders. Future investigation of the interactions between RhoGEF
109
proteins and essential synaptic proteins and ASD-risk genes will be essential to our understanding
of how RhoGEF signaling pathway contribute to the development of brain-related disorders.

110
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Abstract (if available)

Abstract Approximately 1 in 54 children in the US is affected by autism spectrum disorder (ASD). There is a strong genetic basis for ASD but the risk architecture is very heterogeneous. Genetic studies revealed that accumulative ASD risk genes encode proteins essential in synapse function. And a growing body of evidence shows alteration in synapse function in human patients as well as animal models. Glutamatergic synapses, the most common excitatory synapses, are formed on mushroom-like protrusions, dendritic spines. These dendritic spines are filled up and supported by a dense skeletal meshwork of actin filaments. The small GTPase Rac1, a key regulator of actin polymerization, has been implicated as a convergence point of many ASD risk genes. ❧ Here we have identified a large cluster of ASD mutations in the Rac1 activating domain of Trio. The large number of ASD-related mutations we find in the small domain results in a very high genome-wide statistical confidence that these mutations contribute to ASD. We revealed that the majority of these mutations eliminated Rac1 activation and weakened synapse transmission. One mutation D1368V distinctively enhanced glutamatergic synaptogenesis. Downstream of Trio, we characterized a severe ID-related mutation C18Y in Rac1. The mutation C18Y disrupts GTP-mediated Rac1 activation and prevents the induction of long term potentiation (LTP), the cellular mechanism involved in learning and memory. Upstream of Trio, we found that ASD risk gene Neuroligin-1 regulates synapse function through its interaction with Trio and its paralog Kalirin. We found that ASD-related mutation N1080I in Trio disrupted interaction between Trio and Neuroligin and prevented Neuroligin mediated synaptogenesis. The synaptogenic hyper functional ASD-related mutation Trio D1368V rely on Neuroligin1 signaling to pathologically enhance synaptogenesis. Together our findings point to the “Neuroligin-Trio-Rac1” pathway as a possible key pathway that contribute to the development of ASD-related disorders.

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Asset Metadata

Creator Tian, Chen (author)

Core Title The role of Rho-GEF signaling in synapse function and autism-related disorders

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Degree Conferral Date 2021-12

Publication Date 09/30/2021

Defense Date 06/29/2021

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

Tag AMPA receptor,autism,dendritic spine,glutamatergic synapse,neurodevelopment disorder,NMDA receptor,OAI-PMH Harvest,Rho-GEF

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Advisor Arnold, Don (committee chair), Coba, Marcelo (committee member), Dickman, Dion (committee member), Herring, Bruce (committee member)

Creator Email chentian301@gmail.com,tianc@usc.edu

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