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Nebula/DSCR1 upregulation preserves axonal transport and memory function in a Drosophila model for Alzheimer's disease

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Nebula/DSCR1 upregulation preserves axonal transport and memory function in a Drosophila model for Alzheimer's disease

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NEBULA/DSCR1 UPREGULATION PRESERVES AXONAL TRANSPORT AND
MEMORY FUNCTION IN A DROSOPHILA MODEL FOR ALZHEIMER’S DISEASE

By

Jillian Lee Satter Shaw

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

May 2015

Copyright 2015 Jillian Shaw

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EPIGRAPH

“Life shrinks and expands in proportion to one’s courage.” ~ Anaïs Nin

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DEDICATION

For my supportive and hysterical family: Lisa, John, and Brian Shaw

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ACKNOWLEDGMENTS

“Whatever course you decide upon, there is always someone to tell you that you are
wrong. There are always difficulties arising that tempt you to believe that your critics are
right. To map out a course of action and follow it to an end requires courage.”

~ Ralph Waldo Emerson

These words have always seemed particularly poignant in reference to the
scientific process and research. As scientists, we are trained to be critical, skeptical, and
to pierce through the barriers of our own limitations in order to ask impossible questions.
In my case, those questions have revolved around understanding the importance and
function of a complicated gene upregulated in both Down Syndrome and Alzheimer’s
disease. I would have thrown up my hands in surrender early on had it not been for my
brilliant and supportive advisor, Dr. Karen Chang, whose insight, drive, and intellectual
creativity has been a source of inspiration for me over the course of my research. From
designing plans of attack to combat tough reviewers on papers to sharing my excitement
for the results of crucial experiments, I will be forever grateful for Karen’s mentorship
and guidance. Trading insights from hot-off-the-press papers and finding ways to design
new experiments to push my research to greater depths made coming to lab an
intellectual adventure.
I would also like to thank the team of accomplished and driven scientists who
made up my Dissertation Committee: Dr. Derek Sieburth, Dr. Jeannie Chen, Dr. Michael
Jakowec, and Dr. Carol Miller. The quality of their questions regarding my research
proposal prepared me for presenting work to a wider scientific audience at conferences,
and I have greatly benefited from their support and scientific discussions. A special thank
you as well to Dr. Cheryl Craft and the William Hansen Sandberg Memorial Foundation

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for honoring my research with the foundation scholar award to benefit neurodegenerative
research. I am incredibly grateful for the Zilkha Neurogenetic Institute and the
community of scientists with whom I’ve been lucky enough to interact. The quality of the
speakers, symposiums, and Alzheimer’s conferences held as a part of this collaborative
research organization have kept me inspired by what ground-breaking insight is possible
through research. I’m grateful to ZNI for providing me with funding through the Zach
Hall Travel Grant in order to travel to New Orleans to present research at the Society for
Neuroscience meeting. In addition, I’d like to thank the Neuroscience Graduate Program
for contributing to my attendance at the Drosophila Genetics Conference in San Diego.
The acknowledgments section would be incomplete without thanking the
members of the Chang and Sieburth labs who stood next to me at the bench day after day
to offer insights and provide rich conversations. Specifically, I am grateful to have
worked alongside Dr. Catherine Brégère, Jooyeun Lee, Kathy Ha, Dr. Shixing Zhang,
Ken Chan, Syed Quadri, Han Wang, Trisha Staab, and Jason Chen.
On a personal note, I would like to thank my family for their endless patience in
listening to my ramblings about data and scientific theories. I cherish our Sunday
morning coffee chats and dinner discussions combining neuroscience, law, and an
abundance of laughter. Words fall short of how grateful I am for your support, love, and
ability to keep me laughing. I became a scientist to answer questions relating to human
disease, and I’m particularly grateful for the weekly reminders that success is dependent
on making one’s own luck through hard work, perseverance, and creativity. Thank you
for helping me see that what we do as scientist really matters; I’m grateful for the
encouragement to not lose sight of how special it is to work on understanding the

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mysteries of how we form memories or the mechanism by which mitochondria are
trafficked. Lastly, thank you to the friends and USC Neuroscience family who inspired
me to climb mountains of the literal and figurative variety. I’m forever in your debt for
the adventures, laughs, nights of music, summit conquerings, Sierra road trips, and
campfire discussions:

Caroline Hudnut, Elizabeth Cole, Jenny Van Wyk, Asya Magazinnik, Kingson Man,
Anna Kamitakahara, Mona Sobhani, Jenny McGrady-Achiro, Natalie Kintz, Panthea
Heydari, Madeline Andrews, Hanke Heun-Johnson, Kate Fehlhaber, Helder Filipe, Glenn
Fox, Margaret Dwyer, Tomoyo Namigata, Vilay Khandelwal, Simren Dulai, Juan
Velazquez, Nick Goeden, Lei Liew, Brad Gasser, Leigh Komperda, Caty Tems, Raina
Pang, Jessica Rathburn, Radhika Palkar, Brian Leung, Colin Flinders, Jennifer Johnson,
Aaron Webman, Vanessa Marx, Justin Kenderes, Mallori Sheets, Katie Newton, Heidi
Tungseth, Jesse Holcomb, Kate Heller, and Bari Turetsky.

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TABLE OF CONTENTS

EPIGRAPH ii
DEDICATION iii
ACKNOWLEDGMENTS iv
LISTS OF TABLES/FIGURES x
ABSTRACT 13
CHAPTER 1: Introduction 15
1.1 The Link between Down Syndrome and Alzheimer’s Disease 15
1.2 RCAN-1, DSCR1, Nebula – A calcineurin inhibitor by any other name 17
1.3 Nebula Upregulation in Drosophila 21
1.4 Axonal Transport Problems in Neurodegeneration 22
1.5 Drosophila Learning and Memory 27
1.6 Summary 32
CHAPTER 2: Nebula/DSCR1 Upregulation Delays Neurodegeneration
and Protects against APP-Induced Axonal Transport Defects 34
2.1 Abstract 34
2.2 Introduction 35
2.3. Materials and Methods 37
2.4 Results 43
2.4.1 Upregulation of Nebula delays APP-induced Neurodegeneration 43
2.4.2 Nebula Upregulation Ameliorates APP-induced Aggregate
Formation in Axons 46
2.4.3 Nebula Enhances Anterograde Transport of Amyloid
Precursor Protein 57
2.4.4 Nebula Restores Anterograde and Retrograde Trafficking of
Synaptic Vesicles and Mitochondria 59
2.4.5 APP Overexpression does not Alter Microtubule Integrity 64
2.4.6 Nebula Mitigates APP-induced Phenotypes by Regulating
Calcineurin and GSK-3β 67

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2.4.7 Complex Interaction between Calcineurin and GSK-3β
Regulates Axonal Transport 74
2.5 Discussion
2.5.1 Summary of Results 76
2.5.2 Insights into Mechanisms Underlying APP Transport Defects 77
2.5.3 Calcineurin and GSK-3β Signaling Interactions 80
2.5.4 Implications for Delayed Progression of AD in DS 81
2.6 Acknowledgments 82
2.7 Supplementary figures 83

CHAPTER 3: Bidirectional regulation of APP-induced memory defects by
Nebula/DSCR1 – a protein upregulated in Alzheimer’s disease
and Down Syndrome 94
3.1 Abstract 94
3.2 Introduction 95
3.3 Materials and Methods 97
3.4 Results
3.4.1 Nebula/DSCR1 Upregulation Rescues APP-mediated
Impairments in Short-Term Memory 102
3.4.2 Nebula Upregulation Protects Against APP-induced
Impairments in Long-Term Memory 105
3.4.3 Nebula Restores APP-mediated Memory Impairments
through Calcineurin Inhibition 107
3.4.4 Co-upregulation of APP and Nebula Exacerbates Short-
Term Memory Impairments and Exacerbates Mitochondrial
Dysfunction 113
3.4.5 Acute Inhibition of Calcineurin Rescues APP-
mediated STM Impairments in Aged Flies 119
3.5 Discussion 122
3.6 Supplementary Figures 123
3.7 Acknowledgments 133

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CHAPTER 4: Summary and Future Directions 134
4.1 Summary 135
4.2 Nebula recues APP-mediated MBO Accumulation in Axons 132
4.3 Nebula rescues APP-mediated Impairments in Cell Signaling 136
4.4 Two-faced Regulation of Memory by Nebula 138
4.5 Future Directions 139
REFERENCES 141

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LIST OF TABLES

Table S3-1. Sensorimotor responses in 2-4 DAE Drosophila 129
Table S3-2. Sensorimotor responses in 42-45 DAE Drosophila 130
Table S3-3. Sensorimotor responses of MB-GeneSwitch4-GAL4 131
Table S3-4. Sensorimotor responses post-calcineurin inhibitor treatment 132

List of Figures

Chapter 1
1.1 Transient expression of RCAN1-1L (DSCR1) promotes
adaptation to oxidative stress 19
1.2 Chronic upregulation of RCAN1/DSCR1 causes long-term
inhibition of calcineurin 20
1.3 Kinesins are regulated by kinases that phosphorylate motor proteins 25
1.4 Signaling pathways linking abnormal kinase and phosphatase
activity to axonal degeneration 26
1.5 Graphical depiction of the activation of CREB 28
1.6 Drosophila Pavlovian olfactory conditioning apparatus 30
1.7 Model for olfactory-based shock avoidance learning in Drosophila
mushroom body neurons 31

Chapter 2
2.1. Nebula overexpression reduces APP-induced degeneration
structurally and functionally 45
2.2 Nebula upregulation rescues APP-dependent aggregate
accumulation in axons 49
2.3. Nebula overexpression restores synaptotagmin level

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and enhances APP delivery to the synaptic terminals 52
2.4 Nebula co-upregulation rescues synaptic bouton over-
proliferation caused by APP 56
2.5 Nebula upregulation enhances anterograde transport of
APP-YFP in larval motor axons 58
2.6 APP upregulation causes defective transport of synaptotagmin
that is restored by Nebula co-upregulation 60
2.7 Nebula alters APP-induced mitochondrial transport defects 63
2.8 APP overexpression does not alter gross microtubule structure
in the axons or NMJ 66
2.9. Nebula modulates APP-dependent phenotypes by restoring
calcineurin signaling 69
2.10 APP upregulation triggers changes in GSK-3ß signaling downstream
of calcineurin and affects synaptotagmin-kinesin interaction 72
S2.1 Levels of APP and Nebula driven by Gmr-GAL4 83
S2.2 Levels of Nebula in the indicated fly lines 84
S2.3 Levels of APP and Nebula in the indicated transgenic lines driven
by the pan-neuronal Elav-GAL4 driver 85
S2.4 Levels of APP and Nebula in the brains of 3
rd
instar larvae 86
S2.5 Nebula reduction decreases synaptotagmin delivery to the
neuromuscular junction (NMJ) and causes locomotor deficits 87
S2.6 Nebula modulates Drosophila APPL-induced transport deficits in a
similar fashion to human APP 88
S2.7 Nebula co-overexpression increases delivery of Fasciclin to the
synaptic terminal 89
S2.8 APP overexpression does not significantly alter distribution of
mitochondria 90
S2.9. Modulation of APP-induced phenotypes by calcineurin 91
S2.10 Calcimycin application increases the fluorescence intensity
of Case12 signal in fly neurons 92
S2.11 Graphical representation of the interactions between APP, Nebula,

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Calcineurin and GSK-3β 93

Chapter 3
3.1 Co-upregulation of APP and Nebula rescues APP-induced STM
Impairments 104
3.2. APP-mediated impairments in long-term memory are rescued by
upregulating Nebula 106
3.3.A APP-mediated impairments in STM are rescued by restoring
calcineurin or PKA activity 108
3.3.B and C APP-mediated impairments in STM are rescued by Nebula’s
capacity to restore PKA and p-CREB 112
3.4.A Co-upregulation of APP and Nebula enhances age-dependent
memory impairments and exacerbates mitochondrial dysfunctions 116
3.4.B and C Co-upregulation of APP and Nebula enhances age-dependent
memory impairments and exacerbates mitochondrial dysfunctions 117
3.4.D Co-upregulation of APP and Nebula enhances age-dependent
memory impairments and exacerbates mitochondrial dysfunctions 118
3.5 Acute inhibition of calcineurin by transient Nebula upregulation or
pharmacological treatment enhances STM in aged flies 121
S3.1 Memory deficits not attributed to differences in APP expression 123
S3.2 Treatment of Drosophila aged 42-45 days with CspA and FK506
effectively inhibits calcineurin activity to levels of young controls 124
S3.3 Age-dependent decline in STM performance in flies with
altered PKA activity 125
S3.4. Reducing calcineurin in the presence of APP preserves STM
performance until 30-33 DAE when is stops protecting 126
S3.5. Human DSCR1 rescues STM performance for flies co-upregulating
APP and DSCR1 127
S3.6. Reducing calcineurin with RNAi strategy does not exacerbate age-
dependent ATP decline to the same extent as upregulating Nebula 128

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ABSTRACT

The Down syndrome critical region 1 gene (also called regulator of calcineurin
and Nebula) is located in the DSCR region of human chromosome 21 and upregulated in
post-mortem brains from Down syndrome (DS) and Alzheimer’s disease (AD) patients.
Questions remain as to whether elevated expression of DSCR1 is exacerbating disease
pathology or delaying degeneration. The goal of this dissertation is to gain insight into
the role of DSCR1/Nebula in Alzheimer’s disease. Over the course of my research, I
explored the functional interaction between DSCR1/Nebula and the amyloid precursor
protein (APP), which is known to cause AD when duplicated or upregulated in DS. I find
that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration, ameliorates
axonal transport defects caused by APP overexpression, and preserves memory function
in young flies by regulating phosphatase and kinase activity.
APP upregulation results in the accumulation of pre-synaptic proteins in the axons
and increases in calcineurin and glycogen synthase kinase 3 beta (GSK-3β) activity.
Live-imaging experiments reveal that nebula facilitates the transport of synaptic proteins
and mitochondria affected by APP upregulation. Impaired transport of essential
organelles caused by APP perturbation is thought to be an underlying cause of synaptic
failure and neurodegeneration in AD, these findings imply that correcting calcineurin and
GSK-3β can prevent APP-induced pathologies.
I further investigate the effects of DSCR1/Nebula upregulation on amyloid
precursor protein-induced learning and memory deficits. I find that while upregulation of
nebula alone impairs memory, co-upregulation of APP and nebula can effectively restore
APP-induced memory defects, but only in young Drosophila. Nebula/DSCR1

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overexpression rescues perturbations in calcineurin and cAMP signaling caused by APP
overexpression. Surprisingly, nebula accelerated age-dependent memory impairments,
increased reactive oxygen species, and enhanced mitochondrial dysfunction in aged flies.
This finding suggests that upregulation of DSCR1 may delay APP-induced memory
problems initially but enhances memory decline at a later age. My data further
demonstrates that acute upregulation of Nebula/DSCR1 is neuroprotective in the presence
of APP upregulation; however, chronic upregulation of nebula negatively affects
memory. Finally, I present evidence for calcineurin inhibition as a novel target for
therapeutic intervention in preventing axonal transport and memory impairments
associated with AD.

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Chapter 1

Introduction

1.1 The link between Down syndrome and Alzheimer’s disease
By the time individuals with Down syndrome reach 40 years of age, virtually all have
developed sufficient plaques and neurofibrillary tangles for a diagnosis of Alzheimer’s
disease (Cenini et al. 2014). Down syndrome arises from a triplication of chromosome 21
that contributes to developmental changes in the brain that cause intellectual disability.
Of the genes localized to chromosome 21, amyloid precursor protein (APP) is the most
likely culprit contributing to the accelerated onset of Alzheimer’s in this population.
Mutations in the APP gene are sufficient to cause familial Alzheimer’s disease (Lahiri et
al. 2005) and it is thought that increases in APP result in increased production of ß-
amyloid (Aß) – the misfolded component of toxic extracellular plaques seen in post-
mortem Alzheimer’s brains. A case has been made for intracellular Aß contributing to
Alzheimer’s pathology in Down syndrome brains (Domenico et al. 2013). The
Alzheimer’s pathology of Down syndrome patients is unique from sporadic AD patients
as Aß plaques begin to accumulate in DS brains in individuals as young as 8 (Leverenz et
al. 1998). This plaque accumulation accelerates with aging and increases throughout the
lifetime. Despite differences in the age of onset, the pattern and immunohistochemical
staining of the neurofibrillary tangles and senile plaques observed in DS patients are
similar to the pathology seen in AD patients (Lemere et al. 1996).
In early work by Wisniewski et al., it was determined that almost all post-mortem
brains from Down syndrome patients older than 40 had AD neuropathology (Wisniewski

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et al. 1985). A parallel study found that the average age of onset of dementia for this
population is between 50 and 55 years (Prasher and Krishnan 1993) with 40% of Down
syndrome patients being diagnosed with dementia by 60 years of age (Coppus et al.
2006). Consistent with this finding, two additional clinical studies evaluating dementia in
Down syndrome patients reported that no individuals between the ages of 20 and 29 had
dementia (Franceschi et al. 1990; Prasher and Chung 1996; Prasher and Filer 1995).
Reports from clinical studies such as these suggest a period of protection despite the
increased risk for Alzheimer’s.
Despite the presence of AD neuropathology in almost all DS adults older than 40,
there exists a subset of the population that never develop the clinical signs of dementia.
Dementia is assessed in Down syndrome patients independently from the initial cognitive
impairments associated with developmental problems. DS individuals display symptoms
that are similar to Alzheimer’s patients in the general population. By 30 years of age,
28% of Down syndrome patients have severe cognitive decline characterized by apraxia
and agnosia (Oliver et al. 1998). Apraxia is the inability to initiate a learned behavior
despite understanding what behavioral or motor output is required. Agnosia is impaired
sensory processing and object recognition that is independent of memory loss. Early
stages of dementia are indicated by a decline in verbal abilities. Severity of dementia is
thought to have increased when Down syndrome patients lose social skills and display
changes in personality and behavior. These more severe indicators of dementia are
thought to reflect frontal lobe damage and executive function irregularities (Cooper et al.
1998). 75% of individuals with Down syndrome will develop dementia as compared to

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13% of age-matched controls (Head et al. 2012) implying a genetic link between DS and
dementia.
Although dementia manifests earlier in DS patients than in the general population
relative to the early development of AD neuropathology in DS, there is actually a delay
between the development of degenerative pathology and cognitive dysfunction. Given
this observation, it is thought that compensatory protective mechanisms may be
responsible for this disparity between the severity of neuropathology and delay of
dementia. A greater understanding of the cell signaling changes that delay dementia in
the presence of neuropathology may provide insight into sporadic forms of Alzheimer’s
as well as therapeutic approaches for delaying dementia in DS patients.
The focus of this dissertation is exploring the genetic interaction between APP and
DSCR1 and the effect upregulating these genes has on axonal transport, cell signaling,
and memory performance using Drosophila melanogaster as a model organism.

1.2 RCAN-1, DSCR1, Nebula – A calcineurin inhibitor by any other name
Patients with a partial trisomy of chromosome 21 have enabled the identification
of the DS region on chromosome 21 that contributes to phenotypes associated with Down
syndrome. Down syndrome is the most common genetic cause of mental retardation and
is clinically associated with congenital heart disease, immune system impairments, facial
dysmorphology, and the early onset of Alzheimer’s disease. DS affects approximately 1
in 700 live births. Mysteries remain as to exactly what role DSCR1/RCAN1 is playing in
the brain. The DSCR1 gene is located in the DS region and is expressed in the central
nervous system and the heart. The interest surrounding this gene revolves around the

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observation that it is upregulated in fetal and adult Down syndrome brains as well as
post-mortem brains from AD patients (Antonarakis et al. 2006). DSCR1/RCAN1 belongs
to a highly conserved calcipressin family with the capacity to bind to and inhibit
calcineurin. Calcineurin is a Ca
2+
/calmodulin serine/threonine phosphatase known to
regulate cell signaling pathways critical for learning and memory (Klee et al. 1979;
Kingsbury and Cunningham 2000; Fuente et al. 2014). The Drosophila ortholog of
DSCR1 is called Nebula; the nebula protein shares 64% similarity in its amino acid
sequence with human DSCR1 and 43% identity (Chang and Min 2003).
RCAN1-1L is the isoform of RCAN1/DSCR1 predominately expressed in the
human brain (Ermak et al. 2012; Ermak et al. 2013). DSCR1 with its capacity to inhibit
calcineurin is involved in regulating downstream targets important for stress responses to
reactive oxygen species, learning and memory, axonal transport, tau
hyperphsophorylation, the mitochondrial adenine nucleotide transporter (ANT), and the
ADP/ATP exchange in mitochondria (Chang and Min 2003; Ermak et al. 2013; Chang
and Min 2005; Chang et al. 2011). RCAN1 is transiently upregulated in response to
oxidative stress and stress attributed to calcium dyshomeostasis. In Drosophila, nebula
binds to the mitochondrial ANT and regulate the transport of ADP and ATP. ANT plays
a role in the mitochondrial permeability transition pore (mtPTP) that is crucial for
apoptotic and necrotic cell death (Zorov et al. 2009). Individuals with Down syndrome
exhibit higher levels of reactive oxygen species in their brains in comparison with brains
from the general population. Oxidative damage is increased in pre-natal Down syndrome
brains compared to non-DS controls (Odetti et al. 1998). Oxidative damage is thought to
exacerbate the progression of Alzheimer’s in Down syndrome.

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DSCR1 has been implicated as being in the Down syndrome candidate region and
as an adaptive response shock gene that can be transiently upregulated in response to
oxidative stress (Ermak et al. 2001). The impact of increasing DSCR1 varies depending
on the level of stress. Transient elevation of DSCR1 under acute stress has been shown to
be neuroprotective. Prolonged elevation of DSCR1 is associated with the
neurodegeneration associated with Alzheimer’s disease and Down syndrome.

Figure 1.1 Transient expression of RCAN1-1L (DSCR1) promotes adaptation to
oxidative stress while chronic upregulation may contribute to cell death and degeneration
(Ermak et al., 2013).

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Figure 1.2 Chronic upregulation of RCAN1/DSCR1 causes long-term inhibition of
calcineurin potentially contributing to increased neurodegeneration through
mitochondrial defects and aberrant kinase signaling (Ermak et al., 2013).

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1.3. Nebula Upregulation in Drosophila
Previous research performed by my mentor, Dr. Karen Chang, determined that
overexpression of three genes located on chromosome 21, dap160/itsn1, synj/synj1, and
nla/dscr1, influences synaptic morphology, vesicle recycling, and locomotor movement
(Chang and Min 2009). Specifically, dap160 overexpression was associated with aberrant
subcellular distribution of synaptojanin and nebula upregulation resulted in changes in
the phosphatase activity of synaptojanin. Syanptojanin is a target of calcineurin and is a
known phosphatidylinositol phosphatase important for endocytosis. Calcineurin can
dephosphorylate synaptojanin and stimulates its activity. This research laid important
groundwork for understanding Down syndrome phenotypes at the neuronal level. Down
syndrome brains are known to have altered shape, number, and density of synapses. In
systematically investigating how gene overexpression influenced synapses, this work
suggested that restoring levels of gene expression has the potential to reduce synaptic
defects seen in DS brains.
Expanding on the investigation into the role of DSCR1/nebula in contributing to
learning and memory impairments associated with Down syndrome, Dr. Chang generated
mutants of nebula. Both the nebula loss-of-function mutant and overexpressors displayed
severe learning impairments attributed to imbalances in biochemistry rather than
alterations in the morphology of the brain. The findings from this study suggest that
precise regulation of calcineurin signaling plays a crucial role in regulating learning and
memory.

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1.4. Axonal Transport Deficits in Neurodegeneration
The events that precede the accumulation of plaques and tangles associated with
Alzheimer’s disease are of particular interest as therapeutic targets due to the potential to
intervene before progression of the disease accelerates. Two abnormalities that precede
cell death in neurons include synaptic dysfunction and dying-back degeneration of axons
(Kanaan et al. 2013). While axonal transport is often implicated in neurodegeneration, the
link between impairments in fast axonal transport (FAT) and memory decline remains
unexplained. My work seeks to offer evidence for how aberrant activation of signaling
pathways that regulate motor/cargo interactions could negatively regulate microtubule-
based anterograde and retrograde transport. The proper delivery of pre-synaptic proteins
and mitochondria to the synapse as well as the correct distribution throughout the axon is
crucial for neuronal function. In this dissertation, I make a case for normalizing kinase
and phosphatase signaling in order to prevent fast axonal transport deficits and ultimately
delay degeneration.
Post-mortem analysis of Alzheiemer’s brains reveals the existence of dystrophic
axons that contain abnormal accumulations of membrane-bound organelles and
cytoskeletal deficits (Dessi et al. 1997; Praprotnik et al. 1996). The build-up of proteins
in the axons is thought to create “traffic-jams” in the axon that impede the delivery of
synaptic proteins to the terminal. This finding is interesting for identifying abnormalities
in the axons that affect transport before the formation of tau tangles and the accumulation
of amyloid ß plaques. Mammalian and Drosophila models of familial Alzheimer’s
disease confirm that mutations in or upregulation of APP cause synapse loss before the
onset of neuropathology and memory deficits occur (Stokin et al. 2008; Gunawardena

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and Goldstein 2001; Rodrigues et al. 2012). It is worthwhile to explore other mechanisms
that contribute to AD including impaired axonal transport, microtubule abnormalities,
increased oxidative stress, imbalances in calcium signaling, and mitochondrial deficits
(Bamburg et al. 2009).
Identifying the molecular changes that precede the accumulation of Alzheimer’s
pathology and memory impairments is a crucial area of inquiry for intercepting disease
progression. In patients with mild cognitive impairments preceding dementia associated
with Alzheiemr’s, magnetic resonance imaging and diffusion tensor imaging
demonstrated axonal degeneration in the parahippocampal gyrus – a region of the brain
important for connecting neurons in the entorhinal cortex to the dentate gyrus (Stoub et
al. 2006). These hippocampal regions are some of the first affected in Alzheimer’s
disease. Imaging techniques have revealed that axonal degeneration and loss of synaptic
connectivity are present in early stages of the disease. White matter degeneration in the
frontal and temporal lobes, the corpus callosum, and cholinergic system corresponds to
deficits in cognitive function and working memory (Rogalski et al. 2009). Research
evidence points to degeneration of basal forebrain cholinergic neurons (BFCNs) in
contributing to cognitive deficits associated with Alzheimer’s disease and Down
syndrome. In experiments with the Ts65Dn mouse model of Down Syndrome, it was
found that increasing APP caused a decrease in NGF retrograde transport (Salehi et al.
2006). These studies combined point to clinical relevance for evaluating axonal transport
problems as a contributing factor to synapse dysfunction in disease states.
One of the challenges of modeling familial Alzheimer’s disease using invertebrate
and vertebrate models is that no model accurately replicates all aspects of degeneration.

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The value of using Drosophila with their relatively easy to manipulate genomes is the
ability to upregulate a gene such as APP to identify dysfunctional signaling pathways.
Previous work in mice found that expression of pathogenic forms of APP triggers early
abnormalities in synapse function and axonal swellings that precede cell death, amyloid
plaques, and tau hyperphosphorylation (Gilley et al. 2012; Bell et al. 2006). Trafficking
within neurons is complicated by long dendrites and axons that require molecular
components to be synthesized and packaged into membrane bound organelles (MBO’s)
in the neuronal cell body and delivered to the synapse for neuronal survival (Morfini et
al. 2009). Mitochondria and synaptic vesicle precursors are transported by kinesin in the
anterograde direction down to the synaptic terminal. Kinesin exists as a heterotetramer
that contains two kinesin heavy chains or kinesin-1 and two kinesin light chains (KLCs).
The kinesin heavy chain binds to microtubule tracks and performs ATP hydrolysis thus
enabling the mechanochemical activity that enables MBO cargoes to move by the kinesin
holoenzyme (Kanaan et al. 2013) (depicted in Figure 1.3). The kinesin light chain allows
for kinesin to bind to specific membrane bound organelle cargoes (Stenoien et al. 1997).
Retrograde transport is of crucial importance for neuronal survival as it delivers MBOs
containing degradation products, trophic factors, and lysosomes from the axon and
provides delivery to the cell body. Retrograde transport is dependent on cytoplasmic
dynein – a multi-subunit complex that contains two dynein heavy chains and various
other protein subunits that target the DHC to specific cargoes (Susalka et al. 2002).

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Figure 1.3 Kinesins are regulated by kinases that phosphorylate motor protein
subunits and regulate transport activity. The binding of the kiensin heavy chain to the
microtubule track is inhibited by c-Jun-N-terminal kianse 3 (JNK3) and p38α-mediated
phosphorylation. Glycogen synthase kinase 3 (GSK3) in contrast can phosphorylate the
kinesin light chain and enhance dissociation of the membrane bound organelle cargoes
from the motor. Retrograde transport is also influenced by aberrant kinase and
phosphatase activity as phosphorylation of dynein can alter retrograde fast axonal
transport (Kanaan et al., 2012).

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Figure 1.4 Signaling pathways linking abnormal kinase and phosphatase activity to
axonal degeneration, axonal transport defects, and cell death and
neurodegeneration. Alzheimer’s disease is characterized by accumulation of beta
amyloid and tau protein; however, abnormalities in axonal transport, and synapse
dysfunction may precede the events of neurodegeneration.

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Drosophila have successfully been used to model neuorodegenerative diseases that
feature fast axonal transport problems. Experimental evidence indicates that upregulating
Aβ, APP, and human tau all result in impairments in anterograde and retrograde transport
(Gunawardena and Goldstein 2001; Mudher et al. 2004). In mammalian systems, mice
expressing mutant presenilin-1 (another mutation implicated in Alzheimer’s disease) had
severe impairments in mitochondria, specific Trk receptors, APP, synaptophsin, and
syntaxin (Lazarov et al. 2007). An incredibly promising study made a case for linking
enhanced axonal transport with brain plasticity. The researchers uncovered that
environmental enrichment in the APPswe/PS1∆ mouse model of familial Alzheimer’s
resulted in a reduction in tau hyperphosphorylation and increased expression levels of
kinesin subunits (Hu et al. 2010). The interpretation of this study is that brain plasticity
has the potential to ameliorate toxic pathways induced by pathogenic forms of APP
through enhanced axonal transport.

1.5. Drosophila Learning and Memory
The cell signaling pathways underlying memory formation are highly conserved
across species. A transcription factor crucial for activating the expression of genes
necessary for long-term memory formation is cAMP response element binding (CREB).

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Figure 1.5 Graphical depiction of the activation of CREB highlighting its role as a
downstream target of cAMP and Ca
2+
signaling pathways (Kida et al. 2014).

29

Axonal transport is not the only process that is subject to regulation by aberrant
kinase and phosphatase activity. Drosophila learning and memory pathways are highly
dependent on regulation by phosphorylation. Genetic screens have identified Drosophila
mutants with impaired memory to pinpoint the signaling pathways involved. Memory is
frequently assessed in flies using Pavlovian olfactory conditioning (Figure 1.5): olfactory
cues are paired with an aversive electric shock to test avoidance behavior. Short-term
memory (STM) is observed immediately after training and is protein synthesis
independent. STM is dependent on cAMP signaling. Two learning mutants dunce
(encoding a cAMP-specific phosphodiesterase) and rutabaga (encoding an adenylyl
cyclase) indicated that flies with defective cAMP metabolism had abnormal synapse
formation, axonal branches, and neurotransmitter release (Guan et al. 2011). The
amnesiac mutant encodes a gene product for a neuropeptide that stimulates cAMP
activity – further implicating this pathway in regulating learning. Drosophila long-term
memory is dependent on cAMP dependent protein kinase (PKA) activation of CREB
(cAMP response element binding protein) and the resulting gene expression (Pradeep et
al. 2011). Long-term memory can be induced by spaced training: Drosophila are trained
through olfactory conditioning with rest intervals in between training sessions and tested
24-hours later (Tully and Quinn 1985). Inhibition of CREB blocks long-term memory in
Drosophila. CREB activation is dependent on phosphoryation of the Ser133 site raising
questions as to how changing the activity of kinases and phosphatases known to be
aberrantly regulated in Alzheimer’s disease might influence Drosophila learning and
memory.

30

Figure 1.6 Drosophila Pavlovian olfactory conditioning apparatus. Drosophila
learning in a Pavlovian olfactory conditioning test is dependent on NMDA receptors.
(Tischmeyer et al. 1999).

31

Figure 1.7 Model for olfactory-based shock avoidance learning in Drosophila
mushroom body neurons. Phosphorylation of the transcriptional activator CREB by
PKA enables binding to cAMP responsive element (CRE) that enables formation of long-
term memories. When PKA is upregulated for a short period of time, downstream
changes in K+ channels of the axon influences output from the post-synaptic neuron
(Sokolowski 2001).

32

1.6 Summary

Down syndrome is the most common genetic cause of mental retardation and post-
natal developmental abnormalities. Triplication of chromosome 21 results in the
overexpression of the genes localized to that chromosome – amyloid precursor protein
being of particular interest. Almost all individuals with DS develop the pathology
characteristic of Alzheimer’s disease by their 40’s or 50’s; however, not all DS patients
develop dementia or Alzheimer’s disease. Post-mortem brain tissue of DS and AD
patients reveals an upregulation of the Down Syndrome Critical Region 1 (DSCR1) and
APP genes. In this dissertation, I examine how the Drosophila homolog of Down
syndrome critical region 1, nebula, plays a role in ameliorating the transport defects,
aggregate accumulations, and memory impairments associated with APP upregulation. I
propose a neuroprotective role for DSCR1 upregulation as a response to the toxic insults
of cellular changes triggered by APP upregualtion.
Nebula with its ability to inhibit the phosphatase activity of calcineurin, a
Ca2+/calcmodulin dependent serine/threonine phosphatase, down-regulates the GSK3β
pathway that exacerbates Alzheimer’s neuropathology. Axonal transport defects are
thought to precede the onset of Alzheimer’s disease. Proteins encoded by genes linked to
AD can perturb transport; over-expression of amyloid precursor protein exacerbates the
accumulation of vesicles and organelles in the axons. The resulting blockade in transport
impacts the levels of presynaptic proteins that reach the synaptic terminal. Deficits in
kinesin-1 the molecular motor involved in anterograde transport produce a phenoptype
that parallels the aggregate accumulation seen in APP. Kinesin-1 phosphorylation is

33

thought to lead to premature dropping of cargo in the axon. Surprisingly, my research has
suggests that nebula as an inhibitior of calcineurin positively influences downstream
targets that are involved in exacerbating AD axonal transport defects.
The functional link between enhanced axonal transport and preserved memory
function has not been well established. Using genetic approaches, biochemistry, and
behavioral analysis, I demonstrate that co-upregulating APP and nebula reverses the
short-term and long-term memory deficits associated with APP upregulation. I report the
novel finding that Drosophila upregulating APP show an age-dependent increase in
calcineurin activity. Acute pharmacological inhibition of calcineurin using known
inhibitors rescues age-dependent memory impairments in both control flies and those
upregulating APP. This finding suggests that nebula has a bi-modal means of regulating
cell-signaling changes triggered by APP. In young Drosophila, acute upregulation of
nebula confers protection against memory impairments by rescuing PKA and CREB
signaling. In aged flies, however, Nebula exacerbates APP-mediated memory
impairments most likely by contributing to mitochondrial defects and increases in
reactive oxygen species. My dissertation work seeks to answer the question of how
Nebula upregulation regulates APP-induced cell signaling changes. I provide mechanistic
insights into how calcineurin inhibition could influence axonal transport and memory
formation and make a case for acute calcineurin inhibition as a potential therapeutic
target for ameliorating age-dependent memory impairments.

34

Chapter 2

Nebula/DSCR1 Upregulation Delays Neurodegeneration and Protects against APP-
Induced Axonal Transport Defects

Jillian L. Shaw, Karen T. Chang

2.1 Abstract

Post-mortem brains from Down syndrome (DS) and Alzheimer’s disease (AD) patients
show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its
contribution to AD is not known. To gain insight into the role of DSCR1 in AD, we
explored the functional interaction between DSCR1 and the amyloid precursor protein
(APP) which is known to cause AD when duplicated or upregulated in DS. We find that
the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates
axonal transport defects caused by APP overexpression. Live-imaging revels that Nebula
facilitates the transport of synaptic proteins and mitochondria adversely affected by APP
upregulation. Furthermore, we show that Nebula upregulation protects against axonal
transport defects by restoring calcineurin and GSK-3ß signaling altered by APP
overexpression. By rescuing kinase and phosphatase signaling, Nebula preserves cargo-
motor interactions. Impaired transport of essential organelles is an underlying cause of
synaptic dysfunction and neurodegeneration in AD. The experiments presented in this
dissertation indicate that Nebula has the capacity to correct calcineurin and GSK-3ß
signaling impairments and prevent APP-induced declines in function. I propose that
upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation

35

and that calcineurin inhibition may be a novel target for therapeutic intervention in
preventing axonal transport impairments associated with AD.

2.2 Introduction
Alzheimer’s disease (AD) is a debilitating neurodegenerative disease
characterized by gradual neuronal cell loss and memory decline. Importantly, Down
syndrome (DS) individuals over 40 years of age almost always develop
neuropathological features of AD, although most do not develop dementia until at least
two decades later. These findings suggest that DS and AD may share common genetic
causes and that a neuroprotective mechanism may delay neurodegeneration and cognitive
decline. It has been shown that the amyloid precursor protein (APP), which is associated
with AD when duplicated and upregulated in DS, is a key gene contributing to AD
pathologies and axonal transport abnormalities. Here, using fruit fly as a simple model
organism, the role of Down syndrome critical region 1 (DSCR1), another gene located on
chromosome 21 and upregulated in both DS and AD, is examined in the context of
modulating APP phenotypes. Upregulation of DSCR1 (Nebula in flies) is neuroprotective
in the presence of APP upregulation. Nebula overexpression delays the onset of
neurodegeneration and transport blockage in neuronal cells suggesting that signaling
pathways downstream of DSCR1 may be potential therapeutic targets for AD.
Virtually all Down syndrome (DS) adults develop progressive neurodegeneration
as seen in Alzheimer’s disease (AD) and overexpression of the amyloid precursor protein
(APP), a gene located on chromosome 21, is thought to contribute to AD in DS
(Antonarkis et al. 2006; Reeves et al. 2001; Wisniewski and Rabe 1986). Consistently,

36

duplication of a normal copy of APP is sufficient to cause familial AD (Rovelet-Lecrux
et al. 2006; Sleegers et al. 2006), confirming that it is a key gene in AD neuropathologies
seen in DS. This well-known connection between AD and DS provides a unique
opportunity to identify the genetic and molecular pathways contributing to AD. In
addition to APP, another gene likely to play a crucial role in both AD and DS is the
Down syndrome critical region 1 gene (DSCR1, also known as RCAN1) (Ermak et al.
2001). Intriguingly, post-mortem brains from AD patients show increased DSCR1 both at
mRNA and protein levels (Cook et al. 2005; Sun et al. 2011). Studies have also shown
that oxidative stress and Aβ42 exposure can induce DSCR1 expression (Ermak et al.
2002). DSCR1 is located on human chromosome 21 and encodes a highly conserved
calcineurin inhibitor family called calcipressin (Fuentes et al. 1995; Fuentes et al. 2000;
Görlach et al. 2000; Rothermel et al. 2000). DSCR1 has been implicated paradoxically in
both promoting cell survival in response to oxidative stress and in inducing apoptosis
(Kingsbury et al. 2000; Chang and Min 2003; Sun et al. 2011; Porta et al. 2007; Sobrado
et al. 2012). The role of DSCR1 in AD thus remains unclear and an important question is
whether DSCR1 contributes to AD or plays a role in combating the toxic effects of APP
overexpression.
To determine the role of DSCR1 in modulating APP-induced phenotypes, we
used Drosophila as a model system, which has been used successfully to investigate
various human neurodegenerative diseases including AD, Parkinson’s, and
polyglutamine-repeat diseases (Warrick et al. 1998; Feany and Bender 2000; Steffan et
al. 2001; Chen and Feany 2005; Iijima et al. 2004; Clark et al. 2006; Muhammad et al.
2008; Carmine Simmen et al. 2009). Overexpression of APP in both fly and mouse

37

models have previously been shown to cause age-dependent neurodegeneration and
axonal transport defects (Gunawardena and Goldstein 2001; Salehi et al. 2006; Torroja et
al. 1999; Stokin et al. 2005). Furthermore, impaired transport of essential organelles and
synaptic vesicles caused by APP perturbation is thought to be an underlying cause of
synaptic failure and neurodegeneration in AD (Kanaan et al. 2013; Morfini et al. 2009;
Stokin and Goldstein 2006). However, mechanisms for how APP induces transport
defects remain unclear. Here, we show that Nebula, the fly homolog of DSCR1, delays
neurodegeneration and reduces axonal transport defects caused by APP overexpression.
We report that Nebula enhances anterograde and retrograde axonal trafficking as well as
the delivery of synaptic proteins to the synaptic terminal. We find that APP upregulation
elevates calcineurin activity and GSK-3β signaling, but Nebula co-upregulation corrects
altered signaling to restore axonal transport. Together, our results indicate that
Nebula/DSCR1 is neuroprotective in the presence of APP overexpression and further
suggests that Nebula/DSCR1 upregulation may delay AD progression. In addition, our
results for the first time link defective calcineurin signaling to altered axonal transport
and imply that restoring calcineurin and GSK-3β signaling may be a feasible strategy for
treating AD phenotypes caused by APP upregulation.

2.3 Materials and Methods
Fly Stocks
Flies were cultured at 25ºC on standard cornmeal, yeast, sugar, and agar medium
under a 12 hour light and 12 hour dark cycle. The following fly lines were obtained from
the Bloomington Drosophila Stock Center: Gmr-GAL4, UAS-APP695-N-myc (6700),

38

sgg
1
/FM7a, UAS-sgg
S9A
(Sgg constituitively active), UAS-nla-RNAi (27260), UAS-CaNB-
RNAi (27307), UAS-syt.eGFP (6925), UAS-APP.YFP (32039), and UAS-mitoGFP. Elav-
GAL4 stock was generously provided by Dr. Feany (Harvard University), UAS-nla
t1
, and
nla
1
flies were reported previously (Chang and Min 2003). UAS-∆CaN
Act
construct
(constituitively active calcineurin) was generated by deleting the autoinhibitory domain
of the CaNA gene Pp2B-14D and subcloned into the pINDY6 vector similar to that
described (Sullivan and Rubin et al. 2002). UAS-Case12 was generated by inserting
Case12 (from Evrogen) into pINDY6 vector (Souslova et al. 2007). Transgenic flies were
generated by standard germline transformation method (Montell et al. 1985).

Histology
Adult Drosophila of 0, 15, 30, and 45 days of age were collected, decapitated, and
had their proboscis removed. Heads were incubated in Mirsky’s fixative for 30 minutes,
washed with PBS, and post-fixed in 4% paraformaldehyde for 20 minutes. Fly heads
were then transferred to 25% sucrose overnight at 4°C and were subsequently embedded
in Tissue-Tek O.C.T Compound for cryostat sectioning (10 µm). Photoreceptor axons
were immunostained with 24B10 (1:10 Developmental Studies Hybridoma),
Phosphorylated APP (1:400; Sigma), and 4G8 (1:500; Signet).

Phototaxis
Flies were placed in 2 clear round bottom test tubes joined at the opening. After
allowing 2 minutes for the flies to acclimate to the tubes, flies were lightly tapped and the

39

percentage of flies that moved toward light in horizontal position within 30 seconds was
counted.

Immunocytochemistry
Wandering 3
rd
instar larvae were dissected in cold calcium-free dissection buffer
and fixed with 4% paraformaldehyde in PBS for 25 minutes at room temperature (RT).
Samples were blocked in 5% normal goat serum in PBS+0.1% triton for 1 hour at RT and
then incubated with primary antibodies overnight at 4°C. Antibodies included
synaptotagmin (1:1,000; gift from H. Bellen), mAb 4G8 (1:1,000; Signet), ß-tubulin
(1:1,000; DSHB), acetylated tubulin (1:500; Abcam), Cy3-conjugated HRP (1:200;
Jackson Immuno Research). Alexa-conjugated secondary antibodies were applied at
1:500 and samples mounted in Prolong Gold Antifade reagent (Invitrogen).

Static Image Acquisition and Quantification
Images of motor axons and synaptic terminals from NMJ 6/7 in segment A2 or
A3 were captured in a z-series using Zeiss LSM5 scanning confocal. The number of
aggregates was determined manually by counting the number of punctate staining with
intensity above background and size greater than 0.2 µm
2
. For quantification of antibody
staining intensities at the NMJ, dissected larvae were stained together using the same
condition. Images were captured in a z-series and parameters were set to minimize
saturation of pixel intensity. Intensity of Z-projected images was analyzed using ImageJ
and fold change calculated by comparing to the control.

40

Live-Imaging
Wandering 3
rd
instar larvae expressing APP-YFP or GFP-SYT in combination
with other transgenes were dissected in calcium free dissection buffer: 128 mM NaCl,
1mM EGTA, 4 mM MgCl
2
, 2 mM KCl, 5mM Hepes, and 36 mM sucrose. Live-imaging
of GFP-SYTwas done as described (Kuznicki and Gunawardenta 2010). For imaging of
mito-GFP dissected larvae were bathed in HL-3 solution (Louie et al. 2008). Time-lapse
images were acquired at 5-s intervals using a Zeiss LSM5 confocal using minimum laser
intensity to prevent photobleaching and damage to the tissues. Images were acquired for
5 minutes with a 63x lens and a zoom of 1.7. All live-imaging experiments were
completed within 15 minutes starting from the time of dissection in order to ensure health
of the samples.
Live Imaging Analysis
The manual tracking Plugin in ImageJ was used to track individual vesicle and
mitochondria movement. At least 10 frames (>50s) were used to calculate the average
speed of movement. Percentage of movement was determined by counting the percentage
of moving vesicles over the imaging period. A vesicle is labeled as moving if it moved in
three consecutive frames (over a 15-s period) over a distance of at least 0.1µm. Direction
of movement is determined by direction of net displacement of the vesicle at the start of
imaging. Average speed was determined by tracking a vesicle for an uninterrupted run in
either the anterograde or retrograde direction. The total distance of movement was
divided by the total duration of movement in a specific direction. Student’s t-test was
used to determine statistical significance.

41

Line Crossing Locomotor Assay
Deficits in larval locomotor behavior were assessed as described previously
(Chang and Min 2009). Briefly, larvae were washed with PBS and placed in 60 mm petri
dish filled with 1% agarose. Using a moistened paintbrush, 3
rd
instar larvae were
collected and allowed to habituate for 30 seconds. The number 0.5 cm
2
boxes entered was
counted for a 60-s period.

Western Blots
Drosophila adults (1-2 days) were collected on dry ice. Heads were removed and
homogenized in cold RIPA buffer. The brains of 3
rd
instar larvae were dissected and
collected on dry ice. Equal amounts of protein per genotype (10-20 µg) was run on SDS
polyacrylamide gel and transferred to nitrocellulose membrane. Blocking for non-
phosphorylated antibodies was performed using 5% BSA in PBS+0.1% tween (PBS-
TW). Blocking for non-phosphorylated antibodies was done using 5% milk in PBS-TW
for one hour at RT. Membranes were incubated with the following primary antibodies
overnight at 4°C: N-APP (1:5,000; Sigma), ß-tubulin (1:500; Developmental Studies
Hybridoma Bank), Nebula (1:7,000), Fasciclin II (1:50; Developmental Studies
Hybridoma Bank), acetylated tubulin (1:1,000; Cell Signaling), phospho-GSK-3ß Ser9
(1:1,000; Cell Signaling), phospho-GSK-3ß Tyr126 (1:1,000; Cell Signaling), and GSK3
α/β (1:2,000; Cell Signaling). Secondary antibodies used were: anti-mouse Alexa 680
(Invitrogen), anti-rabbit Dylight 800 (Piercenet), anti-mouse coupled HRP or anti-rabbit
coupled HRP. HRP signals were detected using ECL Reagents (GE Healthcare). Alexa
680 and Dylight 800 signals were detected using Odyssey Imaging system (LI-COR

42

Biosciences). For reprobing, membranes were stripped using Reblot Plus strong antibody
stripping solution (Millipore) and reprobed. NIH Image J software was used to measure
signal intensity, and the fold change in specific protein level was normalized to a loading
control and compared to control flies.

Calcineurin Activity
Fly heads were collected over dry ice, decapitated, and homogenized in lysis
buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.02% Sodium Azide). Calcineurin
phosphatase activity was determined using the Ser/Threonine Phosphatase assay kit
(Promega) following the manufacturer’s protocol as previously described (Chang and
Min 2003). 5 µg of protein per genotype was used.

Immunoprecipitation
Drosophila heads were collected on dry ice by passing through molecular sieves
and homogenized in lysis buffer (10 mM HEPES, 0.1 M NaCl, 1% NP-40, 2 mM EDTA,
50 mM NaF, 1 mM NA
3
VO
4
) plus complete Mini protease inhibitor cocktail (Roche).
Lysates were pre-cleared by incubating fly extract with magnetic A/G beads (Thermo
Scientific) for 1 hour at 4°C. Pre-cleared extract was then used for IP using GFP antibody
conjugated to magnetic beads (MBL International). Western blot analysis using an
antibody against the kinesin light chain (1:200; Novus Biologicals) was used to confirm
interaction. To determine the efficiency of GFP pull down, an antibody against GFP
(1:1,000; Abnova) was also used. To eliminate signal contamination form IgG, we used

43

HRP conjugated TrueBlot anti-rabbit IgG (1:1,000; ebioscience) that is specific for native
IgG as secondary antibody.

2.4 Results
2.4.1 Upregulation of Nebula Delays APP-Induced Neurodegeneration
To examine the role of DSCR1 in modulating APP-induced neurodegeneration
and axonal transport defects, we generated transgenic flies containing UAS-APP (APP) in
the presence or absence of UAS-nebula (nla
t1
) (Chang and Min 2003). Targeted
expression of human APP in the fly eyes using the Gmr-GAL4 driver caused age-
dependent degeneration of the photoreceptor neurons, consistent with a previous report
(Greeve et al. 2004). As seen in Fig. 2.1.A, staining with an antibody specific for the
photoreceptor neurons (24B10) and antibody against the APP protein (6E10) revealed the
presence of vacuoles in the retina (arrow). Surprisingly, overexpression of nebula
together with APP (APP;nla
t1
) reduced neurodegeneration as determined by calculating
the fold change in the percentage of area lost), suggesting that Nebula upregulation is
neuroprotective (Figs. 2.1.A and 2.1.B). By 45-days of age, flies expressing both nebula
and APP started to show increased vacuole formation, but the extent of degeneration was
significantly reduced compared to that of APP overexpression, further implying that
Nebula delays the onset of neurodegeneration rather than completely preventing it.
To confirm that Nebula indeed protects against neurodegeneration caused by APP
upregulation, we expressed APP in nla
1
, a previously characterized nla hypomorphic
mutant (Chang and Min 2003). Note that because nebula null alleles are lethal, nebula
hypomorphs were examined (Chang and Min 2003). Fig. 2.1 shows that decreasing

44

Nebula level enhanced APP-induced neurodegeneration in the retina (APP;nla
1
), thus
highlighting the importance of endogenous Nebula protein in conferring neuroprotection.
We did not detect significant neurodegeneration in nla
1
mutant and nla overexpression
even by 45 days of age (data not shown), indicating that APP is necessary for the
observed phenotype. In addition, mitigation of photoreceptor degeneration by Nebula
upregulation is not due to altered expression level of APP, since UAS-LacZ transgene was
included to balance the number of transgenes (we found Gmr-GAL4 is particularly
sensitive to number of transgenes). The level of APP protein in each fly line is also
confirmed by staining with the 6E10 antibody (Fig. 2.1.A) and Western blot analyses
(Fig. S2.1). Comparable level of APP was detected in all transgenic lines, suggesting that
rescue by nebula overexpression is not due to altered APP level.

45

Figure 2.1. Nebula overexpression reduces APP-induced degeneration structurally
and functionally. (A) Cryostat section of 15-day old flies. Neurodegeneration is seen as
holes in the fly retina (arrow). Photoreceptor neurons were detected with mAb24B10
(red) and N-APP antibody (green). To normalize the number of transgenes found in
different fly lines, control, APP overexpression (OE), or APP;nla
1
flies also carry one
copy of UAS-LacZ gene driven by Gmr-Gal4. (B) Fold change in % area lost. n > 4 heads
per genotype and age. (C) Percentage of flies that moved toward light. n = 3-4 separate
tests, total > 100 flies per genotype. All values are mean ± S.E.M, * p≤ .05 compared to
controls, ** p<.05 compared to the indicated genotypes. (D) Sections of 45 day old fly
heads stained with mAb6E10 (APP) and with antibody specific for pT668-APP. White
arrow highlights vacuoles and yellow arrowhead points to aggregates. The medulla in
which R7-8 terminate is magnified on the right. More pT668-APP is seen in the axon
terminals of the photoreceptor neurons (highlighted by magenta arrow).

46

We next determined if Nebula rescues functional defects in photoreceptors by
measuring the ability of flies to see light. Flies are normally phototactic and will move
toward light when placed in test tubes with light source on the opposite end (Benzer
1967). We find that the severity of the vacuole phenotype was paralleled by impairments
in phototactic behavior (Fig. 2.1.C). Flies overexpressing APP showed age-dependent
declines in phototaxis that is delayed by APP and nebula expression (Fig. 2.1.C). Taken
together, these results imply that nebula overexpression protects neurons structurally as
well as functionally against the toxic effects of APP overexpression.

2.4.2 Nebula Upregulation Ameliorates APP-Induced Aggregate Formation in Axons
In addition to influencing photoreceptor degeneration, APP overexpression
caused the formation of APP aggregates in the phororeceptor axons as detected with the
6E10 antibody (Fig. 2.1.D). Previous studies have shown that APP phosphorylated on
threonine 668 (pT668-APP) is preferentially transported in axons (Muresan et al. 2005),
we thus further monitored the distribution of pT668 APP. We found that overexpression
of APP led to pT668-APP accumulation in the photoreceptor axons, whereas APP and
nebula co-overexpression significantly enhanced the delivery of pT668-APP to synaptic
terminals in the medulla (Fig. 2.1.D). These results suggest that APP overexpression may
lead to blocked transport that is alleviated by Nebula.
Axonal transport abnormalities are thought to precede the onset of AD (Stokin et
al. 2005), and APP overexpression has been shown to cause synaptic vesicle accuulations
indicative of blocked axonal transport (Gunawardena and Goldstein and 2001). We thus
further investigated the role of Nebula in modulating APP-induced vesicle aggregation in

47

larval motor axons, which is an excellent system for monitoring vesicle transport because
of the long axons and stereotypical innervation of the neuromuscular junction (NMJ). As
seen in Fig. 2.2.A, APP overexpression in neurons using the Elav-GAL4 driver caused
synaptic vesicle accumulation as detected by synaptotagmin staining in the motor axons,
suggesting abnormal vesicle transport. Staining using the 4G8 antibody to detect APP
revealed that APP aggregates frequently co-localized with synaptotagmin aggregates,
implying that synaptotagmin and APP are either comparably inhibited by physical
blockades within the nerve or that they are transported together as suggested by recent
reports (Groemer et al. 2011; Kohli et al. 2012). Co-upregulation of APP and Nebula
significantly prevented APP-induced synaptotagmin and APP accumulations. Decreasing
Nebula by crossing it into nla
1
background increased the number of synaptotagmin and
APP increased the number of synaptotagmin and APP aggregates slightly, although not
significantly (Fig. 2B). As nla
1
only reduces Nebula level by about 30% and that nla null
alleles are lethal, we used RNAi strategy to further decrease Nebula level (Fig. S.2.2).
Figs. 2.2.A-2.2.B show that greater reduction in Nebula level using the UAS-nla-RNAi
transgene (RNAi-nla) further exacerbated the APP-induced aggregation phenotype. To
ensure that the observed rescue in phenotype is not due to altered APP overexpression,
we monitored the level of neuronal APP protein, as well as Nebula, in different fly lines.
As seen in Fig. S.2.3, APP level was unaltered in flies containing different number of
transgenes, and Nebula manipulations in APP overexpression background showed the
expected changes. Similar results were obtained when performing western blot analyses
using brains dissected from 3
rd
instar larvae (Fig. S2.4). Together, these results confirm
that rescue of APP phenotype by Nebula is not due to altered levels of APP expression.

48

In addition, we examined the effect of altering Nebula levels alone on vesicle
accumulations. Manipulations of Nebula levels alone did not cause synaptotagmin
aggregate accumulation in nerves, suggesting the observed phenotype is APP dependent
(Figs. S.5.A and S.5.B).

49

Figure 2.2 Nebula upregulation rescues APP-dependent aggregate accumulation in
axons. (A) Images showing 3rd instar segmental nerves stained with the indicated
antibodies. Arrowhead points to an example of aggregate found in axon. APP and
synaptotagmin (SYT) aggregates frequently co-localize. (B) The number of SYT and
APP aggregates in axons. N ≥ 10 experiments. * p ≤.05 compared to control, ** P≤0.05
compared to control and APP overexpression. (C) Synaptotagmin aggregates in the
segmental nerves of 3
rd
instar larvae with APPL upregulation. (D) Quantification of the
number of SYT aggregates. N ≥ 4 independent experiments. All values represent mean ±
S.E.M, * p ≤0.05 compared to control, ** P≤0.05 compared to control and APPL
overexpression. Scale bars = 10 µm.

50

To verify that the synaptotagmin aggregate accumulation phenotype is not due to
a non-specific effect of expressing human APP, we also monitored the effect of Nebula
on modulating endogenous fly Appl gene function. Fig. 2.2C shows that upregulation of
APPL in neurons also caused synaptotagmin accumulation in axons. Nebula co-
upregulation significantly reduced the number of synaptotagmin aggregates, whereas
nebula reduction using RNAi significantly exacerbated the phenotype (Figs. 2.2.C and
2.2.D). Together our results support earlier findings that mammalian APP and Drosophila
APPL are functionally conserved (Luo et al. 1992), and further indicate that APP and
APPL-induced axonal transport defects are regulated by Nebula in a similar fashion.
To determine to what degree aggregate accumulation corresponded to altered
delivery of synaptic proteins to the synaptic terminal, we evaluated the levels of both
synaptotagmin and APP in the NMJ. As demonstrated in Figs. 2.3.A-3B, APP
upregulation significantly reduced the level of average synaptotagmin intensity in the
synapse while nebula co-overexpression enhanced the delivery of both synaptotagmin
and APP to the synaptic terminal. This change is not due to altered overall syanptotagmin
or APP levels (Figs. S.2.4 and S.2.5C). Note that the 4G8 antibody does not detect
endogenous fly APPL; therefore, we normalized the level of APP delivered to the
synapse to flies overexpressing APP and nebula. We found Nebula reduction did not
further reduce the amount of synaptotagmin reaching the terminal (Fig. 2.3B), albeit it
did increase the number of APP-induced aggregates in the axon (Fig. 2.2B). This result
indicates that either retrograde transport of synaptotagmin is altered, or the increase in
aggregate number has not yet reached a critical threshold for further impairment. In
addition, although no detectable synaptotagmin aggregate was seen in flies with Nebula

51

reduction alone, a decrease in synaptotagmin staining was detected in the synapse (Figs.
S.2.5B and S5D). This result suggests that either retrograde transport of synaptotagmin is
altered or the increase in aggregate number has not yet reached a critical threshold for
further impairment. In addition, although no detectable synaptotagmin aggregates were
seen in flies with Nebula reduction alone, a decrease in synaptotagmin was detected in
the synapse (Figs. S.2.5B and S.2.5D). This result suggests that Nebula itself may be
required for reliable axonal transport.

52

Figure 2.3. Nebula overexpression restores synaptotagmin level and enhances APP
delivery to the synaptic terminals. (A) Representative images of NMJ staining for the
indicated genotypes. Scale bars = 10 µm. (B) Levels of SYT and APP in NMJ normalized
to flies overexpressing APP;nla
t1
. N ≥ 5 independent experiments. (C) Larval locomotor
activity assay. N = 10 experiments. All values represent mean ± S.E.M, * p ≤0.05
compared to control, **p<0.05 compared to control and APP overexpression.

53

We also examined the effect of abnormal aggregate accumulations and reduced
delivery of synaptic proteins on locomotor behavior. Overexpression of APP dramatically
impaired larval movement (Fig. 2.3C and Movie S.2.1). Nebula co-overexpression
significantly rescued this locomotor defect, in further support of the hypothesis that
Nebula upregulation exerts beneficial effects on synaptic functions by alleviating
abnormal aggregate accumulations. Note that further reduction of Nebula in APP
overexpression background did not significantly worsen the locomotor defect of APP
overexpressing larvae, perhaps due to a threshold effect. Reducing Nebula alone was
sufficient to induce a mild defect in locomotor activity (Fig. S.2.5E), suggesting delivery
of synaptic proteins to the synaptic terminal is crucial for synaptic function.
Similar to APP overexpression, upregulation of APPL decreased the delivery of
synaptotagmin to the synapse. APPL and Nebula co-upregulation showed a higher level
of synaptotagmin in the NMJ, confirming Nebula interacts genetically with APPL to
rescue impairments in transport (Fig. S.2.6A and S6B). We also found that similar to
RNAi-nla larvae, Appl null mutant (Appl
d
) displayed a slight decrease in the level of
synaptotagmin at the synapse independent of aggregate accumulation (Figs. 2.S.6B and
S6C). Reducing Nebula in neurons of Appld larvae with the RNAi-nla transgene driven
by the pan-neuronal nSyb-GAL4 driver (Appld;RNAi-nla/nSyb-GAL4) did not further
enhance the phenotype, suggesting that the two proteins act in the same pathway to
modulate axonal transport.
While monitoring synaptotagmin levels at the NMJ, we also noticed that APP
overexpression triggered changes in synaptic morphology as previously reported (Torroja
et al. 1999; Ashley et al. 2005). Fig. 2.4 shows presynaptic terminals stained with HRP to

54

outline the presynaptic terminals, which revealed an increase in the total number of
boutons and satellite boutons brought upon by APP overexpression. Nebula co-
upregulation also rescued APP-induced synapse proliferation phenotype, but not the
number of satellite boutons (Fig. 2.4.B and 2.4.D). Manipulating levels of Nebula alone
without APP did not influence bouton number or morphology, suggesting that the
satellite bouton phenotype is dependent on the presence of APP in the synapse. Since
reducing Nebula levels alone decreased the delivery of synaptotagmin to the synaptic
terminal without altering synaptic morphology, axonal transport problems are not
secondary consequences of altered synaptic morphology. A plausible mechanism by
which Nebula suppresses the APP-induced over-proliferation phenotype is that Nebula
co-upregulation restores the delivery of proteins required for normal synaptic growth
such as Fasciclin II (FasII), a cell adhesion molecule shown to influence synaptic
morphology. Previous reports suggest that changes in FasII levels differentially affect
synaptic growth and that increasing FasII levels presynaptically can significantly
suppress the increase in bouton number observed in APPL overexpression synapses
(Schuster et al. 1996; Schuster and Davis 1996). We therefore quantified FasII levels in
the NMJ (Fig. S7). We found that overexpression of APP reduced the level of APP
reduced the level of FasII in the NMJ, whereas APP and nebula co-overexpression
restored it (Fig. 2.S.7). While APP upregulation may play other roles in synapse
formation, these results together with previous reports imply that depletion of FasII in the
presynaptic terminal could partially contribute to the hyper-growth phenotype.
Furthermore, our data reveal that Nebula upregulation is effective in protecting against
multiple phenotypes caused by APP overexpression, including age dependent

55

photoreceptor neurodegeneration, vesicle accumulation in axons, and changes in synaptic
morphology.

56

Figure 2.4 Nebula co-upregulation rescues synaptic bouton over-proliferation
caused by APP. (A) Images representing the neuromuscular junction of 3
rd
instar larvae
at segment A2 of muscle segment 6/7. HRP staining outlining synaptic boutons. Scale bar
= 10 µm. (B) Quantification of the number of boutons normalized to the muscle surface
area (MSA). (C) Magnified images demonstrating the presence of satellite bouton
phenotype (arrowheads) in genotypes with APP upregulation. Scale bar = 5 µm. (D)
Quantification of the percentage of boutons that are satellite. n≥6 experiments in (B) and
(D). All values represent mean ± S.E.M, * p≤0.05 compared to control.

57

2.4.3 Nebula Enhances Anterograde Transport of Amyloid Precursor Protein
To directly evaluate the effect of Nebula on APP transport and to determine
whether the observed axonal aggregates correspond to defective axonal transport, we
performed live-imaging of APP tagged with yellow fluorescent protein (APP-YFP). APP-
YFP vesicles in larval motor axons displayed movement in both the anterograde and
retrograde directions over the 2-minute imaging period as represented by kymographs
depicting distance traveled and time in the x- and y- directions, respectively (Fig. 2.5.A).
Nebula co-overexpression had a mild, but significant effect on APP-YFP movement.
Nebula co-upregulation increased the percentage of anterograde moving vesicles and
resulted in reduced number of stationary APP-YFP; knockdown of Nebula using RNAi
increased the number of stationary APP-YFP (Figs. 2.5.A and 2.5.B). Quantification of
the average speed of APP-YFP movement revealed that overexpression of nebula also
increased the speed of APP-YFP movement in both the anterograde and retrograde
directions (Fig. 2.5C). Together, these results suggest that Nebula upregulation enhances
the transport of APP, consistent with the decreased aggregate accumulations of APP in
axons and increased APP staining in the NMJ when Nebula is co-expressed (Figs. 2.2.A
and 2.3.A).

58

Figure 2.5 Nebula upregulation enhances anterograde transport of APP-YFP in
larval motor axons. (A) Representative kymographs depicting trafficking of APP-YFP
vesicles. Stationary vesicles are seen as vertical lines and anterograde movement is
depicted as diagonal lines moving from left to right. Scale bars: 5 µm (X) and 30 seconds
(Y). Red lines in the lower boxed region highlight anterograde moving vesicles. Blue
lines depict retrograde moving vesicles. (B) Quantification of the percentage of
anterograde and retrograde moving vesicles, as well as stationary vesicles in the indicated
genotypes. nebula overexpression significantly increased the relative number of
anterograde moving vesicles. (C) Speed of APP-YFP vesicles in the anterograde and
retrograde directions. Nebula upregulation enhanced the speed in the anterograde and
retrograde directions. All values represent mean ± S.E.M, * p≤0.05 compared to control,
** P<0.05 compared to the indicated genotype. n≥6 independent experiments per
genotype.

59

2.4.4. Nebula Restores Anterograde and Retrograde Trafficking of Synaptic Vesicles
and Mitochondria
To further confirm that Nebula facilitates synaptic vesicle movement in the
presence of APP and to better assess the role of endogenous Nebula in regulating
transport, we also monitored synaptotagmin movement in the motor axons of larvae
expressing GFP-tagged synaptotagmin (GFP-SYT). We find the movement of GFP-SYT
to be highly dynamic with anterograde, retrograde, and bi-directional movement (Fig.
2.6.A). Overexpression of APP dramatically reduced the percentage of vesicles moving
in both the anterograde and retrograde directions while nebula co-overexpression
significantly facilitated synaptotagmin transport in both directions (Figs. 2.6.A and 6B),
albeit retrograde transport was more effectively restored by Nebula. Reducing Nebula
using RNAi further diminished APP-induced synaptotagmin transport in both directions,
confirming interactions between Nebula and APP. Reduction in the overall movement
was also accompanied by a decrease in anterograde and retrograde velocity (Fig. 2.6C).
Together, these results suggest that APP overexpression slows down the overall
movement of vesicles that may lead to accumulation of transported proteins. Nebula co-
overexpression with APP partially restores the defect by increasing the movement and
speed of transport in both the anterograde and retrograde directions.

60

Figure 2.6 APP upregulation causes defective transport of synaptotagmin that is
restored by Nebula co-upregulation. (A) Kymographs depicting movement of GFP-
SYT. Scale bars: 5 µm (X) and 60 s (Y). Red lines: anterograde movement; blue lines:
retrograde movement. (B) Quantification of the total number of moving vesicles, as well
as relative movement in the anterograde and retrograde directions normalized to the total
number of observed vesicles. APP overexpression (OE) caused significant defects in the
movement of GFP-SYT. (C) Speed of GFP-SYT movement in the anterograde and
retrograde directions. Values represent mean ± S.E.M, * p≤.05 compared to control, **
P<0.05 compared to the indicated genotypes. N≥6 independent experiments per
genotype.

61

To understand the role of endogenous Nebula in axonal transport, we examined
the effect of Nebula manipulations on GFP-SYT movement in the absence of APP
overexpression. We find that Nebula upregulation alone did not significantly influence
transport; decreasing Nebula through RNAi was sufficient to reduce the number of
moving synaptotagmin vesicle in both directions, as well as the speed of anterograde
transport (Fig. 2.6). This result is consistent with the decrease in synaptotagmin staining
in the NMJ seen in static images, and further confirms that Nebula is required for
efficient transport of synaptic proteins.
To further determine if general axonal transport is affected by APP and Nebula
upregulation, we also monitored mitochondrial transport. Proper distribution of
mitochondria is vital for normal cell functions and defects in mitochondrial transport can
adversely affect cell survival (Mattson et al. 2008; Iijima-Ando et al. 2012; Iijima et al.
2009). Time-lapse live-imaging was performed in larvae with GFP-targeted to
mitochondria (mito-GFP) for the indicated genotypes (Fig. 2.7). APP upregulation
severely impaired the movement of mitochondria in both the anterograde and retrograde
directions both in terms of percent in motion and the speed of movement (Figs. 2.7.B and
2.7.C). Nevertheless, the APP-induced mitochondrial transport defect was partially
restored by Nebula co-upregulation (Fig. 2.7), similar to what was observed for synaptic
vesicle transport. Manipulations in the level of Nebula did not significantly alter the
overall mitochondrial movement, except that nebula overexpression alone seemed to
enhance both the proportion and the speed of mitochondria transported in the retrograde
direction. This result is consistent with our observation that Nebula co-upregulation was

62

more effective in restoring retrograde GFP-SYT transport. Together, our results suggest
that Nebula influences general axonal transport that extends beyond synaptic proteins.

63

Figure 2.7 Nebula alters APP-induced mitochondrial transport defects. (A)
Kymographs showing mito-GFP movement in motor axons. Scale bar: 5 µm (X) and
60 s (Y). Red lines: anterograde movement; blue lines: retrograde movement. (B)
Quantification of the percent of mobile mitochondria, and mitochondria moving in
the anterograde and retrograde directions. (C) Speed of mitochondrial movement. All
values represent mean ± S.E.M, * p≤0.05 compared to control, ** P<0.05 compared
to the indicated genotypes. n≥6 independent experiments per genotype.

64

Mitochondria are dynamic organelles whose distribution is tightly regulated to meet
the energy demands within the polarized neuron (Shapasan et al. 2012). We find that
despite the decrease in mitochondrial movement in flies overexpressiong APP, the
distribution and density of mitochondria within the proximal axon where imaging was
performed did not vary across genotypes (Fig. S.2.8A). These data imply that impaired
synaptic vesicle transport is not likely caused by local depletion of mitochondria within
the axon. Furthermore, mitochondria did not accumulate near the site of synaptotagmin
aggregate formation in the axons (Fig. S.2.8.B), suggesting that mitochondria are either
able to move past the stalled synaptic vesicle accumulations or that mitochondria travel
on other non-blocked microtubule tracks.

2.4.5 APP Overexpression does not Alter Microtubule Integrity
Despite increasing evidence linking defective trafficking of pre-synaptic proteins,
mitochondria, and signaling molecules to neuropathologies of AD, mechanisms for how
APP overexpression affects axonal transport remain unclear. We first tested the
possibility that APP upregulation impairs axonal transport by influencing overall
microtubule integrity. To this end, we stained the nerves and NMJs with antibodies
against acetylated tubulin, ß-tubulin, and Futsch (Fig. 2.8). Acetylated tubulin is a
marker for stable microtubules (Piperno and Fuller et al. 1985); Futsch is a microtubule
binding protein homolog to human MAP1B and is involved in maintaining microtubule
integrity at pre-synaptic terminals during NMJ outgrowth (Godena et al. 2011). Our data
revealed that APP overexpression did not cause fragmentation of microtubules as
revealed by both acetylated tubulin and ß-tubulin staining in the axons (Fig. 2.8.A), and
filamentous acetylated tubulin staining in the synaptic terminals across all genotypes

65

(Fig. 2.8.B). Note that in Fig. 2.8.B, we also highlighted the presynaptic boutons by HRP
staining (red), since acetylated tubulin in the muscles are also detected in the background.
Western blot analyses of dissected larval brains further confirmed that the overall level of
acetylated tubulin is not altered by APP overexpression (Fig. 2.8.C). Closer examination
of Futsch staining also did not reveal differences in overall microtubule integrity (Fig.
2.8.D). Together, these results suggest that APP overexpression does not cause axonal
transport problems by influencing microtubule stability, which is consistent with a recent
report that showed microtubule stability and acetylated tubulin level in larvae
overexpressing APP-YFP (Weaver et al. 2013).

66

Figure 2.8 APP overexpression does not alter gross microtubule structure in the
axons or NMJ. (A) Images of the larval segmental motor axons stained with
acetylated tubulin (Ac-tubulin) or ß-tubulin as indicated. Microtubule structural
integrity was not influenced by APP overexpression. (B) Representative images of the
larval neuromuscular junction at segment A2 of muscle 6/7 stained with Ac-tubulin
(green) and HRP (red). Background Ac-tubulin signal is also detected in the muscle
(outside the boundaries of HRP staining). (C) Western blot depicting Ac-tubulin
levels for the indicated genotypes. Values were normalized to ß-tubulin,which is used
as a loading control. Lower panel shows quantification of the relative protein level for
the indicated transgene normalized to the control. Values represent mean ± SEM, n =
3 independent experiments. (D) Images of the 3
rd
instar larval NMJ at segment A2 of
muscle 6/7 stained with Futsch (green) and HRP (red). All scale bars = 10 µm.

67

2.4.6. Nebula Mitigates APP-Induced Phenotypes by Regulating Calcineurin and
GSK-3ß

Nebula encodes an inhibitor of calcineurin that is highly conserved across species.
Therefore, the hypothesis that calcineurin inhibition is an underlying mechanism for
Nebula-mediated recue of APP phenotypes was tested. To this end we genetically
altered calcineurin activity in neurons using the UAS-GAL4 strategy. To elevate
calcineurin activity, we expressed a constituitively active calcineurin (CaN
Act
) with its
auto-inhibitory domain deleted (Figs. S.2.9.A and S.2.9.B). To reduce calcineuring
activity, RNAi strategy against the calcineurin B gene (RNAi-CaNB), an obligatory
subunit necessary for calcineurin activity, was used. We find that similar to Nebula
upregulation, decreasing calcineurin using RNAi-CaNB in the presence of APP
significantly reduced synaptotamin aggregate accumulations and synaptic depletion,
as well as restored larval locomotor behavior (Figs. S.2.9C-E). Overexpression of
CaN
Act
together with APP further exacerbated the APP-induced phenotypes (Figs.
2.9A and 2.9.B), whereas co-overexpression of CaN
Act
and nebula diminished the
ability of Nebula to protect against APP-induced transport defects. Similar to larvae
with reduced levels of Nebula (RNAi-nla), larvae expressing CaN
Act
did not show
aggregate accumulations in axons but displayed a reduced level of synaptotagmin
staining in the synapse (Figs. S.2.9.D), indicating active calcienurin overexpression
alone only has modest effect on axonal transport. As shown above, synaptotagmin
aggregate accumulation in nerves and depletion in the synaptic terminals are reliable
indicators of significant transport deficiencies; our results thus indicate that Nebula

68

protects against APP-induced defects through inhibition of calcineurin. Furthermore,
our data present for the first time that APP upregulation influences axonal transport
through activation of calcineurin. This conclusion is further supported by direct
measurement of calcineurin activity, in which we find that APP upregulation
significantly elevated calcineurin activity but is further restored close to normal in
flies overexpressing APP and nebula, or APP and RNAi-CaNB (Fig. 2.9.C).
Overexpression of APP, CaN
Act
, and nebula together showed an intermediate
phenotype in both calcineurin activity and aggregate accumulations, suggesting that
the severity of aggregate accumulation correlated with the level of calcineurin when
APP is upregulated.

69

Figure 2.9. Nebula modulates APP-dependent phenotypes by restoring
calcineurin signaling. (A) Representative images of 3
rd
instar larval mtor axons
stained with synaptotagmin. Arrowheads point to examples of aggregates. Scale bar:
10 µm. (B) Quantification of the number of aggregates found in each genotype. (C)
Calcineurin activity assay. n≥ 10 independent experiments per genotype for all
experiments. (D) Images of 3
rd
instar brain highlighting the imaging area (left).
Magnifed images (right) depict the intensity of the genetically encoded fluorescent
calcium sensor, Case12, across genotypes. (E) Quantification of the relative
fluorescent intensity across genotypes. N = 6 experiments. All values represent mean
± S.E.M. * p≤0.05 compared to control and ** P <0.05 compared to the indicated
genotypes.

70

How does APP upregulation trigger calcineurin activation? Because calcineurin
phosphatase activity is dependent on intracellular calcium concentration (Klee et al.
1979), we examined the possibility that APP overexprssion elevates calcium levels.
Using a genetically encoded fluorescent calcium sensor (Case12) previously shown to
detect calcium with high sensitivity (Chang et al. 2011; Souslova et al. 2007), we
compared Case12 signal across different genotypes. Supplementary data (Fig. S.2.10)
demonstrates that larval brains expressing Case12 displayed a significant increase in
signal following application of calcimycin, a calcium ionophore, comfirming that Case12
construct can indeed detect increases in calcium. Overexpression of APP alone or
overexpression of APP and nebula also caused a significant elevation in Case12 signal in
the larval brain and the ventral ganglion (where the motor neuron cell bodies are located)
as compared to the control (Figs. 2.9.D and 2.9.E). These data imply that an APP-
mediated increase in calcium is triggering the increase in calcineurin activity.
Furthermore, observations that co-overexpression of APP and nebula increased calcium
while simultaneously restoring calcineurin activity indicate that Nebula is influencing
axonal transport through calcineurin inhibition rather than acting at a step modulating
calcium influx.
Mechanisms by which calcineurin regulates axonal transport are not well
understood, but one potential pathway is through regulation of GSK-3ß. Aberrant
activation of GSK-3ß has been associated with AD and calcineurin has been shown to
activate GSK-3ß through dephosphorylation of Ser9 of GSK-3ß in vitro (Hooper et al.
2008; Kim et al. 2009; Hernandez et al. 2013; Mondragon-Rodriguez et al. 2012). It was
suggested that GSK-3ß might negatively influence axonal transport by altering

71

microtubule stability through hyperphosphoryaltion of tau, by inhibiting kinesin light
chain (KLC), or by altering the kinesin motor activity (Mudher et al. 2004; Weaver et al.
2013; Morfini et al. 2002; Sofola et al. 2010). These previous findings led us to
investigate the possibility that Nebula restores APP-dependent transport problems
through calcineurin-mediated regulation of GSK-3ß in vivo. The activity of GSK-3ß is
regulated by phosphorylation and dephosphorylation: dephosphorylation of Ser9 by a
number of phosphatases including calcineurin is required to activate GSK-3ß (Kim et al.
2009; Lochhead et al. 2006), and phosphorylation at Tyr216 site is necessary to enhance
GSK-3ß activity (Peineau et al. 2008; Hughes et al. 1993). Interestingly, phosphorylation
of GSK-3ß at Ser9 can both inhibit GSK-3ß activity and override the increase in activity
even when phosphorylated at Tyr216 (Bhat et al. 2000). Because these phosphorylation
sites are conserved between fly and human, we took advantage of phospho-specific
antibodies to monitor GSK-3ß activity. Western blot analyses using an antibody specific
for phosphorylated Ser9 (pSer9) of GSK-3ß revealed that APP upregulation indeed
reduced the level of pSer9-GSK-3ß while APP and Nebula co-upregulation partially
restored the level to normal (Fg. 10A). This suggests APP upregulation leads to GSK-3ß
activation that is inhibited by Nebula upregulation.

72

Figure 2.10 APP upregulation triggers changes in GSK-3ß signaling downstream of
calcineurin and affects synaptotagmin-kinesin interaction. (A) Western blots
performed using antibody specific for GSK-3ß phosphorylated on Ser9 (pS9-GSK) and
total GSK-3ß. (B) Western blot using antibody specific for GSK phosphorylated at
Tyr214 in Drosophila (pY214-GSK). For (A) and (B) quantification of the ratio of
phosphorylated GSK and GSK-3ß was normalized to control. n≥3 for each, * p<0.05. (C)
Western blots showing reduced interaction between SYT-GFP and kinesin light chain
(KLC). Control indicates parallel immunoprecipitation performed using flies that do not
express SYT-GFP. n=3. *p<0.05. (D) Images of 3
rd
instar larval motor axon stained with
synaptotagmin. Arrowheads highlight aggregates and * indicates background staining
coming from synaptotagmin in the NMJ. Scale bar: 10 µm. (E) Quantification of the
number of aggregates found in each genotype. All values are mean ± S.E.M., * p≤0.05
compared to APP expression. n≥4 independent experiments per genotype.

73

To verify that GSK-3ß activation is due to calcineurin activation, we reduced
calcineurin activity in APP overexpressing flies using RNAi-CaNB (Fig. 2.10.A). We
find that APP and RNAi-CaNB co-overexpression in neurons, which was sufficient to
restore calcineurin activity, completely prevented GSK-3ß dephosphorylation at Ser9
site. This result indicates that APP-induced GSK-3ß dephosphorylation at Ser9 site. This
result indicates that APP-induced GSK-3ß dephosphorylation at Ser9 is dependent on
calcineurin activation in vivo. Note that we did not detect enhanced GSK-3ß
dephosphorylation when APP is expressed together with constitutively active calcineurin
(CaN
Act
) suggesting that calcineurin may in part directly influence the transport through
GSK-3ß-independent pathways.
Our data strongly implicate activation of calcineurin and subsequent GSK-3ß
induction to be a mechanism underlying APP-induced aggregate phenotype. Because
activation of calcineurin alone did not result in synaptotagmin aggregate accumulation,
we further hypothesized that APP upregulation also enhances GSK-3ß activity through
phosphoryaltion at Tyr216. Western blot analyses show that the level of phosphorylated
GSK-3ß at Tyr214 (conserved Tyr216 site in Drosophila) is indeed elevated in flies
overexpressing APP or APP and nebula (Fig 2.10.B). Overexpression of CaN
Act
alone,
however, failed to induce phosphorylation at Tyr214, suggesting that phosphorylation at
Tyr214 is not affected by calcineurin and dependent on the presence of APP. Together,
our data demonstrate that in addition to activating GSK-3ß by reliving inhibition through
calcineurin, APP upregulation further enhances GSK-3ß activity through phosphorylation
at Y214 in fly.

74

Active GSK-3ß has been shown to phosphorylate KLC, leading to detachment of
the cargo from the motor (Morfini et al. 2002; Piginio et al. 2003). Since synaptotagmin
transport was severely inhibited by APP overexpression, and that synaptotagmin
transport can depend on kinesin 3 (Barkus et al. 2008; Pack-Chung et al. 2007) and
kinesin-1 (both KLC and kinesin 1 heavy chain) (Hurd et al. 1996; Gindhart et al. 1998;
Byrd et al. 2001; Toa et al. 2008; Hirokawa et al. 2009), we tested the possibility that
APP overexpression perturbs KLC and synaptotagmin interaction via
immunoprecipitation. APP overexpression indeed reduced synaptotagmin (cargo) and
KLC interaction while overexpression of APP and nebula preserved this interaction (Fig.
10C). These results suggest that Nebula is likely to restore APP-induced axonal transport
defects by correcting GSK-3ß signaling and stabilizing cargo-motor interaction.

2.4.7. Complex Interaction between Calcineurin and GSK-3ß Signaling Regulates
Axonal Transport
Having demonstrated that APP activates calcineurin signaling to regulate GSK-3ß
phosphorylation, we next examined if reducing GSK-3ß can restore axonal transport. In
the presence of APP upregulation, decreasing Shaggy (Sgg; fly homolog of GSK-3ß) in
flies with APP overexpression (sgg
1
;APP) resulted in significant suppression of the APP
aggregate phenotype (Figs. 2.10.D and 2.10.E). This result is consistent with a recent
report demonstrating mild enhancement of APP-YFP movement when GSK-3ß is
reduced (Weaver et al. 2013). Surprisingly, normal calcineurin activity was detected in
these flies (1.00±0.16 fold of control for Sgg
1
;APP vs. (1.75±0.25 fold of control for
APP). This result suggests the existence of feedback regulation of calcineurin activity and

75

further implies that either a change in calcineurin activity or GSK-3ß signaling could be
responsible for the observed rescue. We therefore generated flies expressing APP and
constituitively active calcineurin in sgg
1
background (sgg
1
; APP/CaN
Act
). Note that we
used the hypomorphic allele sgg
1
because sgg null animals are lethal (Ruel et al. 1993).
Consistent with GSK-3ß being downstream of calcineurin, reducing Sgg diminished the
effect of CaN
Act
in enhancing the APP phenotype (Figs. 2.9.B and 2.10.E). We also
expressed the constituitively active Sgg (sgg
S9A
) which surprisingly showed the same
phenotype as APP overexpression. Calcineurin activity assay showed an unexpected
decrease in calcienurin activity (0.74±0.06 fold of control) in these flies, suggesting that
constitutive GSK-3ß activation in the absence of calcineurin activation is sufficient to
disrupt axonal transport potentially through phosphorylation of KLC. Interestingly, we
find that overexpression of the constituitively active Sgg in neurons alone was sufficient
to induce aggregate accumulation similar to flies with APP overexpression (Figs. 2.10.D
and 2.10.E). Calcineurin activity assay revealed that these flies showed an increase in
overall calcineurin activity (1.65±0.30 fold of control). This increase in calcineurin
activity by active Sgg may be due to GSK dependent phosphorylation of Nebula, which
has been shown to cause activation of calcineurin (Takeo et al. 2012). Since over-
activation of calcineurin and GSK-3ß pathway in the absence of APP upregulation fully
replicated the aggregate accumulation phenotype, it suggests that abnormal activation of
both the GSK-3ß and calcineurin pathways are necessary for the severe axonal transport
defect and aggregate accumulation phenotypes.

76

2.5. Discussion
2.5.1 Summary of Results
We have demonstrated a novel role for Nebula, the Drosophila ortholog of
DSCR1, in ameliorating axonal transport impairments associated with the upregulation of
APP. We find that Nebula upregulation significantly delayed photoreceptor
neurodegeneration and dramatically decreased the axonal “traffic jam” phenotype caused
by APP overexpression. Reducing Nebula independent of APP was sufficient to trigger
defects in axonal transport, suggesting that Nebula independent of APP was sufficient to
trigger defects in axonal transport, suggesting that Nebula is normally required for
reliable delivery of synaptic cargos, likely through calcineurin dependent pathways. We
demonstrate for the first time that APP overexpression causes calcineurin-dependent
activation of GSK-3ß kinase in vivo, thus implicating altered calcineurin signaling as a
novel mechanism regulating axonal transport (Fig. S.2.11). We find that co-upregulation
of Nebula preserved the vesicular cargo to molecular motor interaction, ameliorated
axonal transport defects, and protected against locomotor deficits. As impaired transport
of essential organelles and synaptic vesicles caused by perturbation of APP is thought to
precede synaptic failure and neurodegeneration in AD, our findings further suggest that
DSCR1 upregulation may be a neuroprotective mechanism used by neurons to combat
the effects of APP upregualtion and delay progression of AD.

77

2.5.2 Insights into Mechanisms Underlying APP-Transport Defects
Although upregulation of APP has been shown to negatively influence axonal
transport in mouse and fly models, mechanisms by which APP upregulation induces
transport defects are poorly understood. Several hypotheses have been proposed,
including titration of motor/adaptor by APP, impairments in mitochondria bioenergetics,
altered microtubule tracks, or aberrant activation of signaling pathways (Mitchell and Lee
2012). The motor/adaptor titration hypothesis suggests that excessive APP-cargos titrate
the available motors away from other organelles – resulting in defective transport of pre-
synaptic vesicles. The finding that Nebula co-upregulation enhanced the movement and
delivery of both synaptotagmin and APP to the synaptic terminal argues against this
hypothesis. Earlier findings suggest that Nebula upregulation alone impaired
mitochondrial function and elevated ROS level (Chang and Min 2005) implying that
Nebula is not likely to rescue APP-dependent phenotypes by selectively restoring
mitochondrial bioenergetics. Consistent with recent reports showing normal microtubule
integrity in flies over-expressing either APP-YFP or activated GSK-3ß (Weaver et al.
2013), our data revealed normal gross microtubule structure in flies with APP
overexpression. Together, these results suggest that changes in gross microtubule
structure and stability is not a likely cause of APP-induced transport defects.
Instead, our data supports the idea that Nebula facilitates axonal transport defects
by correcting APP-mediated changes in phosphatase and kinase signaling pathways.
First, we find that APP upregulation elevated intracellular calcium level and calcineurin
activity, and that restoring calcineurin activity to normal suppressed the synaptotagmin

78

aggregate accumulation in axons. The observed increase in calcium and calcineurin
activity is consistent with reports of calcium dyshomeostasis and elevated calcineurin
phosphatase activity found in AD brains (LaFerla 2002; Reese and Taglialatela 2011;
Garwood et al. 2013), as well as reports demonstrating elevated neuronal calcium level
due to APP overexpression and increased calcineurin activation in Tg2576 transgenic
mice carrying the APP
swe
mutant allele (D’Amelio et al. 2011; Santos et al. 2009).
Second, APP upregulation resulted in calcineurin dependent dephosphorylation of GSK-
3ß at Ser9 site, a process thought to activate GSK-3ß kinase. APP upregulation also
triggered calcineurin-independent phosphorylation at Tyr216 site, which has been shown
to enhance GSK-3ß activity. The kinases that phosphorylate APP at Tyr216 are currently
not well understood. It will be important to study how APP leads to Tyr216
phosphorylation in its future. Based on our results, we predict that APP overexpression
ultimately leads to excessive calcineurin and GSK-3ß activity, whereas nebula
overexpression inhibits calcineurin to prevent activation of GSK-3ß (Fig. S.2.11). Our
findings that nebula co-overexpression prevented GSK-3ß activation and enhanced the
transport of APP-YFP vesicles are consistent with a recent report by Weaver et al., in
which they find that decreasing GSK-3ß in fly increased the speed of APP-YFP
movement. Furthermore, consistent with our result that APP upregulation triggers GSK-
3ß enhancement and severe axonal transport defects, Weaver et al. did not detect changes
in GFP-synaptotagmin movement in the absence of APP upregulation.
Active GSK-3ß has been shown to influence the transport of mitochondria and
synaptic proteins including APP, although the exact mechanism may differ between
different cargos and motors (Chen et al. 2007; Morel et al. 2010). One mechanism

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proposed for GSK-3ß mediated regulation of axonal transport is through phosphorylation
of KLC1, thereby disrupting axonal transport by decreasing the association of the
anterograde molecular motor with its cargos (Morfini et al. 2002). In agreement with this
result, we find that APP reduced KLC-synaptotagmin interaction while Nebula
upregulation preserved it. Synaptotagmin transport in both the anterograde and retrograde
directions were affected, consistent with previous reports showing that altering either the
anterograde kinesin or retrograde dynein is sufficient to affect transport in both directions
(Brady et al. 1990; Pilling et al. 2006). Our results also support work suggesting that
synaptotagmin can be transported by the kinesin-1 motor complex in addition to the
kinesin 3/imac motor (Barkus et al. 2008; Pack-Chung et al. 2007; Hurd et al. 1996;
Gindhart et al. 1998; Byrd et al. 2001; Toda et al. 2008; Hirokawa et al. 2009). As kinesin
1 is known to mediate the movement of both APP and mitochondria (Muresan et al.
2005; Pilling et al. 2006; Reis et al. 2012; Gunawardena et al. 2013), and that
phosphorylation of KLC has been shown to inhibit mitochondrial transport (De Vos et al.
2000) detachment of cargo-motor caused by GSK-3ß mediated phosphorylation of KLC
may lead to general axonal transport problems as reported in this dissertation. However,
GSK-3ß activation may also disrupt axonal transport by influencing motor activity or
binding of motors to the microtubule tract. Interestingly, increased levels of active GSK-
3ß and phosphorylated KLC and dynein intermediate chain (DIC), a component of the
dynein retrograde complex, have been observed in the frontal complex of AD patients
(Morel et al. 2012). Genetic variability for KLC1 is thought to be a risk factor for early-
onset of Alzheimer’s disease (Andersson et al. 2007). There is also increasing evidence
pointing to the role of GSK-3ß in regulating transport through modulating kinesin

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activity. It is thought that activation of this kinase exacerbates neurodegeneration in AD
through tau hyperphosphorylation (Weaver et al. 2013; Hooper et al. 2008). It will be
interesting to investigate if Nebula also modulates these processes in other
neurodegenerative disease models.

2.5.3 Calcineurin and GSK-3ß Signaling Interactions
Although calcineurin has been shown to regulate many important cellular
pathways, the link between altered calcineurin and axonal transport, especially in the
context of Alzheimer’s disease, has not previously been established. We show that
calcineurin can regulate axonal transport through both GSK-3ß independent and
dependent pathways. This is supported by our observation that the severity of the
aggregate phenotype was worse for flies expressing APP and active calcineurin than it
was for flies expressing APP and active GSK-3ß. These findings point to a role for
calcineurin in influencing axonal transport directly, perhaps through dephosphorylation
of motor or adaptor proteins. Our data also indicate that calcineurin in part modulates
axonal transport through dephosphorylation of GSK-3ß; however, upregulation of APP is
necessary for the induction of severe axonal transport problems, mainly by causing
additional enhancement of GSK-3ß signaling. GSK3 inhibition is widely discussed as a
potential therapeutic intervention for AD. This dissertation suggests that calcineurin
inhibition may be a more effective target for delaying degeneration by preserving axonal
transport.

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2.5.4 Implications for Delayed Progression of AD in DS
DSCR1 and APP are both located on chromosome 21 and upregulated in DS
(Rovelet-Lecrux et al. 2006; Sleegers et al. 2006). Overexpression of DSCR1 alone has
been contradictorily implicated in both conferring resistance to oxidative stress and in
promoting apoptosis (Sun et al. 2011; Ermak et al. 2002; Porta et al. 2007; Sobrada et al.
2012). Upregulation of Nebula/DSCR1 has also been shown to negatively impact
learning and memory in fly and mouse models through altered calcineurin pathways
(Chang and Min 2003; Dierssen et al. 2011). How could upregulation of DSCR1 be
beneficial? We propose that DSCR1 upregulation in the presence of APP upregulation
compensates for the altered calcineurin and GSK-3ß signaling, shifting the delicate
balance of kinase/phosphatase signaling pathways close to normal, therefore preserving
axonal transport and delaying neurodegeneration. We also propose that axonal transport
defects and synapse dysfunction caused by APP upregulation in our Drosophila model
system occur prior to accumulation of amyloid plaques and severe neurodegeneration
similar to that described for a mouse model (Stokin et al. 2005).
DS is characterized by the presence of AD neuropathologies early in life, but most
DS individuals do not exhibit signs of dementia until decades later, indicating that there
is a delayed progression of cognitive decline (Cota et al. 2012). The upregulation of
DSCR1 may in fact activate compensatory signaling mechanisms that provide protection
against APP-mediated oxidative stress, aberrant calcium, and altered calcineurin and
GSK-3ß activity.

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2.6 Acknowledgments
Thank you to Katherine Ha who contributed excellent technical assistance, Dr.
Hugo Bellen for generous donation of the synaptotagmin antibody, the Developmental
Studies Hybridoma bank for antibodies, Dr. Seth Ruffins for consulation on analysis of
live-imaing and microscopy, and Dr. Kyung-Tai Min for critical reading of the
manuscipt.

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2.7 Supplementary Figures

Figure S2.1. Levels of APP and Nebula driven by Gmr-GAL4. (A) Western blots
depicting the levels of APP and Nebula in flies overexpressing the indicated transgenes
using the Gmr-GAL4 driver. Control flies carry one copy of UAS-LacZ driven by Gmr-
Gal4. To normalize the number of transgenes found in different fly lines, UAS-LacZ was
crossed into the background of flies with APP overexpression (OE), or APP;nla
1
flies.
Note that transgenic line nla
t1
contains nebula transgene tagged with HA, and hence the
overexpressed Nebula appears as a high band. (B) Quantification of the relative protein
levels for the indicated fly lines. Values represent mean ± SEM, n = 4 independent
experiments. * p<0.05 compared to control. All calculations were normalized to loading
control, ß-tubulin.

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Figure S2.2 Levels of Nebula in the indicated fly lines. (A) Western blots showing the
level of Nebula in nla
1
mutant (containing one copy of Elav-Gal4 so that it is in the same
genetic background), and RNAi-nla driven by the neuronal Elav-Gal4 driver. (B)
Quantification of Nebula protein level. Values represent mean ± SEM, n = 3 independent
experiments. * p<0.05 compared to control. All calculations were normalized to loading
control, ß-tubulin.

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Figure S2.3 Levels of APP and Nebula in the indicated transgenic lines driven by the
pan-neuronal Elav-GAL4 driver. (A) Western blots depicting the levels of APP and
Nebula in fly heads overexpressing the indicated transgenes using the Elav-Gal4 driver.
Protein loading level is indicated by ß-tubulin. Because transgenic line nla
t1
contains the
nebula transgene tagged with HA, the overexpressed Nebula protein appears as a higher
band. (B) Quantification of APP and Nebula proteins in fly head extracts. Values
represent mean ± SEM, n = 4 independent experiments. * P<0.05 compared to control.
All calculations were normalized to loading control, ß-tubulin.

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Figure S2.4 Levels of APP and Nebula in the brains of 3
rd
instar larvae. (A) Western
blot depicting the level of APP in larvae overexpressing the indicated transgenes. All
transgenes were driven by the neuronal Elav-Gal4 driver. Lower graph shows
quantification of APP protein level in dissected larval brains. Relative values depicted in
comparison to APP. (B) Western blot depicting the level of Nebula in larval brain
extracts. Lower graph shows quantification of Nebula protein level. All values represent
mean ± SEM, n≥3 independent experiments. All calculations were normalized to loading
control, ß-tubulin. * P < 0.05 compared to control.

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Figure S2.5 Nebula reduction decreases synaptotagmin delivery to the
neuromuscular junction (NMJ) and causes locomotor deficits. (A) Synaptotagmin
(SYT) staining in the segmental motor axons. (B) Quantification of STY aggregate
number and protein level in the NMJ. N = 6 independent experiments. (C) Western blots
showing that the level of overall STY was not altered. (D) SYT staining in the NMJ for
the indicated genotypes. Right panel shows pseudo colored SYT staining in the NMJ for
the indicated genotypes. Right panels show pseudo colored SYT staining and intensity
scale. (E) Locomotor assay. n = 10 independent experiments. For (B) and (E), values
represent mean ± SEM * indicates P<0.05 compared to control. Scale bars = 10 µm.

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Figure S2.6 Nebula modulates Drosophila APPL-induced transport deficits in a
similar fashion to human APP. (A) Synaptotagmin (SYT) staining in the NMJ for the
indicated genotypes. The APPL overexpression lines were driven by the pan-neuronal
Elav-Gal4 driver and the Appl
d
;RNAi-nla line was driven by the pan-neuronal nSyb-Gal4
driver. Right panels show pseudo-colored SYT staining and intensity scale. (B)
Quantification of SYT level in the NMJ normalized to the control. Values represent the
mean ± SEM, n = 6 independent experiments * indicates P<0.05 compared to control
unless otherwise indicated. (C) SYT staining in the axonal nerves of the Appl
d
lines.
Scale bars = 10 µm.

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Figure S2.7 Nebula co-overexpression increases delivery of Fasciclin to the synaptic
terminal. (A) Pseudo-colored images (left column) of Fasciclin (FasII) staining in the
NMJ of 3
rd
instar larvae (A2 of muscle 6/7). Right panels show FasII (green) and HRP
staining (red) outlining the synaptic bouton structure. Scale bar = 10 µm. (B)
Quantification of the relative intensity of FasII in the terminal normalized to the control.
Values represent mean ± SEM, n = 6 independent experiments * indicates P < 0.05
compared to control unless otherwise indicated, n ≥ 5 independent experiments per
genotype.

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Figure S2.8 APP overexpression does not significantly alter distribution of
mitochondria. (A) Quantification of the number of mitochondria normalized to the
length of the nerve. (B) APP overexpression did not cause accumulation of mitochondria
near sites of SYT aggregates (red). To determine distribution of mitochondria,
mitochondrial targeted GFP (mito-GFP) was expressed together with the indicated
transgenes. Scale bar = 10 µm. n ≥ 6 independent experiments and all values represent
mean ± SEM.

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Figure S2.9. Modulation of APP-induced phenotypes by calcineurin. (A) Diagram of
the constitutively active calcineurin construct (CaN
Act
) with calmodulin and
autoinhibitory domain (AID) deleted. (B) Calcineurin activity. CaN
Act
indicate flies with
transgene only but no driver, and CaN
Act
OE indicates CaNAct overexpression in
neurons. n = 4 assays. (C) Images showing NMJs stained with SYT (green; bottom
panels). Upper panels are pseudo-colored images with intensity scale shown on the right.
(D) Quantification of SYT level in the NMJ. n>6 independent experiments. (E)
Locomotor activity. n = 10 independent experiments. All values represent mean ± SEM.
* indicates p<0.05 compared to control and ** P<0.05 compared to the indicated
genotype.

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Figure S2.10 Calcimycin application increases the fluorescence intensity of Case12
signal in fly neurons. (A) DIC image of the larval brain (left) highlighting the region
imaged (right). Fluorescence intensity was determined before and after calcimycin
treatment. (B) Quantification of the relative fluorescent intensity before and after
calcimycin addition. n = 3 independent experiments. All values represent mean ± SEM. *
indicates P<0.05 compared to control.

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Figure S2.11 Graphical representation of the interactions between APP, Nebula,
Calcineurin and GSK-3β. (A) The left-hand panel depicts downstream signaling events
arising from APP upregulation. APP overexpression increases intracellular calcium
levels. Calcium influx activates calcineurin (CaN) activity and an unknown pathway that
leads to phosphorylation of GSK-3β at Y216 (or Y214 in Drosophila). Phosphorylation
of GSK-3β at Y216 is required for enhancement of GSK-3β activity. Active calcineurin
also dephosphorylates GSK-3β at Ser9 to relieve inhibition of GSK-3β, leading to
activation of GSK-3β with enhanced activity. GSK-3β activation strongly contributes to
axonal transport problems through downstream pathways, whereas calcineurin
(independent of GSK-3β pathway) weakly contributes to axonal transport problems. APP
upregulation therefore impairs axonal transport through two independent but interacting
signaling pathways. (B) The right hand panel introduces the downstream signaling events
arising from interactions between APP and Nebula co-upregulation. Nebula inhibits
calcineurin activity, thereby preventing the activation of GSK-3β by calcineurin. Even
though APP still triggers GSK-3β phosphorylation at Y216, phosphorylation at Ser9
overrides and inhibits activation of GSK-3β. Nebula inhibition of calcineurin rescues
APP-mediated axonal transport defects by restoring both GSK-3β and calcineurin
activity.

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Chapter 3

Bidirectional regulation of APP-induced memory defects by Nebula/DSCR1 – a
protein upregulated in Alzheimer’s disease and Down syndrome

Jillian L. Shaw, Shixing Zhang, and Karen T. Chang

3.1 Abstract
Down syndrome (DS) individuals exhibit Alzheimer’s disease (AD) pathologies
early in life, but most don’t develop dementia until decades later. The etiology of this
progressive dementia is not known. Here, we show that nebula/DSCR1 (Down syndrome
critical region 1), a protein upregulated in DS and AD, has dual roles in modulating APP
(amyloid precursor protein)-induced memory defects. Using Drosophila as a model, we
find nebula overexprssion initially protects against APP-induced memory defects by
correcting calcineurin and cyclic-AMP signaling pathways, but accelerates the rate of
memory loss and exacerbates mitochondrial function in older Drosophila. We report that
transient upregulation of nebula/DSCR1 or acute pharmacological inhibition of
calcineurin in aged flies reversed APP-induced memory loss, suggesting that calcineurin
dyshomeostasis and chronic nebula/DSCR1 upregulation contribute to age-associated
memory impairments in AD in DS.

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3.2 Introduction
Down syndrome (DS) due to full or partial triplication of chromosome 21 greatly
increases the risk of Alzheimer’s disease (AD). By age 65, approximately 75% of DS
individuals will develop dementia as compared to 13% of age-matched controls (Bush
and Beail et al. 2004). Despite an early presence of the neurochemical changes seen in
AD brains (Leverenz et al. 2004; Perluigi et al. 2014; Head et al. 2001), dementia is
delayed in most DS individuals until after mid-life, suggesting both a genetic risk for
dementia and the existence of a neuroprotective period before the onset of memory
impairments. Mechanisms underlying age-dependent memory decline are poorly
understood. The well-known connection between DS and AD provides a unique
opportunity to identify common genetic factors contributing to AD and age-associated
dementia.
To uncover mechanisms underlying age-dependent memory decline in AD and
DS, we examined the functional interactions between two genes encoded by chromosome
21 and upregulated in both DS and AD. The amyloid precursor protein (App), encoded by
chromosome 21, is a known risk gene for AD since either mutations or duplications of
App is associated with familial AD (Sleegers et al. 2006; Rovelet-Lecrux et al. 2005;
Goate et al. 2006; Murrell et al. 1991; Chartier-Harlin et al. 1991). Studies have shown
that overexpression of wildtype human APP in both mouse and Drosophila models
causes cognitive deficits prior to β-amyloid accumulation (Greeve et al. 2004; Simón et
al. 2009; Charkraborty et al. 2011), suggesting that APP perturbation could contribute to
dementia independent of β-amyloid plaques. Another gene encoded by chromosome
21that is likely to play a crucial role in AD is the Down syndrome critical region 1

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(Dscr1; also known as Rcan-1) gene. Post-mortem brains from DS and AD patients show
an upregulation of DSCR1 mRNA and protein levels (Cook et al. 2005; Sun et al. 2011).
Oxidative stress and β-amyloid exposure have also been shown to induce DSCR1
upregulation (Ermak et al. 2002). DSCR1 encodes an evolutionarily conserved inhibitor
of calcineurin, as erine/threonine calcium calmodulin phosphatase important for
numerous physiological pathways including memory, cell death, and immunity (Fuentes
et al. 1995; Fuentes et al. 2000; Aramburu et al. 2000). Studies have shown that altering
levels of DSCR1 in mouse and Nebula (fly homolog of DSCR1) in Drosophila severely
impaired memory (Chang and Min 2003; Hoeffer et al. 2007; Martin et al. 2012).
However, upregulation of Nebula/DSCR1 has been shown to both promote and inhibit
cell survival following oxidative stress (Ermak et al. 2001; Sun et al. 2011; Porta et al.
2007) as well as protect against APP-induced neurodegeneration and axonal transport
defects (Shaw and Chang 2013). It thus remains unknown how Nebula/DSCR1
upregulation will affect APP-induced memory defects.
The interaction between Nebula and APP has not previously been examined in the
context of learning and memory. Precise regulation of calcineurin signaling is crucial for
learning performance. Overexpression of APP elevates calcineurin activity raising the
question of whether altering this signaling pathway and its downstream targets influences
learning and memory performance in Drosophila. Drosophila melanogaster can be
classically conditioned by association of an aversive odorant with an electric shock (Tully
and Quinn 1985). This method has been used to investigate the neuronal mechanisms
underlying learning and memory pathways. Groups of approximately 60-100 flies were
trained with methyl cyclohexanol (MCH) by pairing this aversive odorant for 60 seconds

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with an electric shock (60 volts). Subsequently, flies were exposed to octanol (OCT) for
60 seconds without the shock. Immediately following training, the flies were given 120
seconds to chose between the two odors in a T-maze. Learning was indicated by
avoidance of the conditioned stimulus (the odor with the electric shock). To evaluate the
effect of co-upregulation of DSCR1 and APP on learning performance, we generated
transgenic flies containing UAS-APP (APP) in the presence or absence of UAS-nebula
(nla
t1
). Tissue specific expression of human APP in the mushroom body by using the
C739-GAL4 driver caused severe impairments in learning. The mushroom body contains
approximately 2500 neurons known as the Kenyon cells. Ablation of mushroom body
structures results in defective associative learning (Davis 2012) indicating that the fly
model can effectively be used to study memory.

3.3 Materials and Methods
D. melanogaster stocks and material
Flies were cultured at 25° on standard cornmeal, yeast, sugar, and agar medium
under a 12-hour light and 12-hour dark cycle. The following fly lines were obtained from
the Bloomington Drosophila Stock Center: UAS-APP695-N-myc (6700), sgg
1
/FM7a,
UAS-nla-RNAi (27260), UAS-CaNB-RNAi (27307), and UAS-PKA-C1.FLAG. The Elav-
Gal4 stock was generously provided by Dr. Mel Feany (Harvard University). The UAS-
nla
t1
was reported on previously as was the C739-GAL4 driver (Chang and Min 2003).
The mushroom body gene-switch flies were kindly provided by Dr. Gregg Roman
(University of Houston).

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Pavlovian Olfactory Learning and Memory Assays
Drosophila aged 2-4, 7-10, 30-33, and 42-45 days were tested in the T-maze for
the Pavlovian olfactory conditioning assay (Tully and Quinn 1985). Prior to testing, the
apparatus was humidified for 2 hours with a vacuum connected to an air bubbler. Flies
were given 6 trials to distribute equally between 3-octanol (OCT) (Sigma Organics) and
4-methylcyclohexanol (MCH) (Sigma-Aldrich) and tube lengths were adjusted
accordingly to reach equal distribution. Groups of ~ 50-100 flies were trained with one
odor (MCH) that was paired with a shock (60 V). Subseqently, flies were given 60
seconds of exposure to a non-shock paired odor (OCT). Immediately following training,
STM was evaluated by giving the flies 2 minutes to distribute between the two odors. To
test long-term memory, flies were subjected to the same training protocol but repeated for
10 training sessions with 15-min rest intervals between sessions. Flies were then tested
immediately and 24-hours post-training. All training was performed under red light in the
dark room. The performance index was calculated as the number of flies that avoided the
unconditioned stimulus (OCT) subtracted from the number of flies that avoided the
conditioned stimulus (odor and electric shock). This value was multiplied by 100 and
divided by the total number of flies. For sensorimotor tests, flies were habituated to the
apparatus and given 2 minutes to choose between the arm with the aversive odor or air.
This was repeated for electric shock and reported as the percentage of flies that avoided
MCH, OCT, and shock.

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PKA Activity Assay
To evaluate PKA activity, fly extracts from Drosophila heads were prepared
according to the conditions of the PepTag Assay for non-radioactive detection of cAMP-
dependent protein kinase (Promega). Briefly, extracts from homogenized Drosophila
heads were incubated in a 16-µl reaction mixture with a fluorescent-tagged A1 peptide
(PKA substrate), reactivation buffer, and protease inhibitor for 30 min. The reactivation
was inactivated by heating at 95°C for 10 min. The non-phosphorylated A1 peptide is +1
charged. Post-phosphorylation by active PKA in the samples it switches to -1 charge.
Using electrophoresis, the phosphorylated and non-phosphorylated forms can be
separated on a 0.8% agarose gel. To visualize the fluorescent tag in the gel, a UV lamp
was used and images captured. Integrated density values were taken for phosphorylated
and non-phosphorylated bands using Image J (National Institutes of Health). The relative
PKA activity in each sample was calculated as the amount of phosphorylated A1 peptide
divided by the total A1 peptide and normalized to the amount of total protein.

Western Blots
Drosophila adults (1-3 DAE) were collected on dry ice and heads were removed
and homogenize in cold RIPA buffer. Equal amounts of protein per genotype (10-20 µg)
was run on SDS polyacrylamide gel and transferred to a nitrocellulose membrane.
Blocking for non-phosphorylated antibodies was done using 5% milk in PBS-Tween for
one hour at RT. Blocking for phosyphorylated antibodies was done using 5% BSA in
PBS + .01% Tween. For detection of phosphorylated CREB, a nuclear extraction was

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performed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo
Scientific). Membranes were incubated in the following antibodies overnight at 4° C: N-
APP (1:2,000; Sigma), ß-Tubulin (1:1,000; Developmental Studies Hybridoma Bank),
Phosphorylated CREB (1:1,000; Cell Signaling), and TATA-BP (1;1,000; AbCam).
Secondary antibodies included anti-mouse and anti-rabbit HRP-coupled. HRP signals
were detected using ECL Reagents (GE Healthcare). For reprobing, membranes were
stripped using Reblot plus (Millipore). NIH Image J software was used to measure signal
intensity. The fold change was then normalized to the loading control and compared to
either control or APP flies.

Calcineurin Activity
Fly heads were collected over dry ice and homogenized in lysis buffer (10 mM
Tris pH 7.4, 1 mM EDTA, 0.02% sodium azide). Calcineurin phosphatase activity was
determined using the Ser/Threonine Phosphatase Assay kit (Promega) following the
manufacturers protocol and previously reported (Chang and Min 2003). 5 µg of protein
per genotype was used.

ATP Abundance
ATP content was assayed using a colorimetric assay kit (BioVision) according to
the manufacturer’s protocol. Briefly, Drosophila heads were collected on dry ice and
homogenized in ATP assay buffer. Samples were deproteinated by centrifuging the
extract for one hour at 14,000 rpm in 10 kDa Vivaspin columns (Sartorius Stedim

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Biotech). Sample were incubated with reaction mixture and imaged with the microplate
reader with an OD of 570 nm.

RU486 Transient Induction
Flies that are either 2-5 days or 30-33 days were fed with or without 500 µM
RU486 in 2% sucrose solution for 2 days before training and testing according to method
described by Mao et al.

Pharmacolgocial Acute Inhibition of Calcineurin
Drosophila adults of 1 day or 42 days were transferred to vials of food containing
the calcineurin inhibitors cyclosporin A or FK506. Drugs were diluted and mixed in the
food to a final concentration of 30 µM of cyclosporine A or FK506 for feeding for 3
days. This concentration was selected as it effectively inhibited calcineurin to control
levels with calcineurin activity assay following drug treatment. Post-feeding Drosophila
adults were tested on the Pavlovian olfactory conditioning test and heads were used for
the calcineurin assay.

ROS detection
Flies were briefly etherized and brains were dissected in Schneider’s medium
(Invitrogen). Freshly dissected fly brains were incubated with 20 µM dihydroethidium
(Invitrogen) at room temperature for 15 mins. After several washes in PBS, brains were
mounted in Prolong Gold Antifade Medium (Molecular Probes) between coverslips. To

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minimize variations in signal intensity, brains of different fly lines were mounted on the
same slide and fluorescence intensities of at least 6 brains were averaged.

3.4 Results
3.4.1 Nebula/DSCR1 upregulation rescues APP-mediated impairments in short-
term memory
Drosophila and humans share conserved cell signaling components and pathways
essential for learning and memory formation, thus providing an effective model system
for studying mechanisms contributing to age-dependent memory impairments (Davis et
al. 2011; Dubnau and Tully 1998). To evaluate the effects of Nebula/DSCR1
upregulation in modulating APP-induced memory impairment, we measured short-term
memory (STM) in Drosophila using the classic Pavlovian olfactory conditioning assay
(Tully and Quinn 1985; Préat 1998). We assayed STM mainly because it is primarily
impaired in the early stages of AD (Klekociuk and Summers et al. 2014; Grady et al.
2001). To bypass the locomotor defects caused by pan-neuronal APP overexpression
(Shaw and Chang 2013), we selectively upregulated APP and Nebula in the mushroom
bodies, structures important for olfactory memory in Drosophila . Consistent with
previous reports, upregulation of either Nebula or APP alone caused severe STM defects
(3-min memory) in young flies between 2-4 days of age (Chang and Min 2005;
Chakraborty et al. 2011), but co-upregulation of Nebula and APP surprisingly restored
STM to normal (Fig. 3.1.A). Western blot analyses and sensorimotor assays confirmed
preservation of STM in flies with APP and Nebula co-overexpression is not due to
differences in transgene expression or changes in sensorimotor responses (fig. S.3.1 and

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table S.3.1). Reducing nebula expression using the UAS-nla-RNAi (RNAi-nla) transgene
also resulted in STM defect alone, as well as when in the presence of APP
overexpression. To evaluate whether fly nebula and human DSCR1 regulated APP-
mediated impairments in memory similarly, human DSCR1 was upregulated in the
presence of APP and shown to also rescue short-term memory (Figure S.3.5). Together,
these results confirm that Nebula/DSCR1 is important for normal STM, and interacts
genetically with APP to protect against APP-induced STM defects.

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Figure 3.1

Figure 3.1 Co-upregulation of APP and Nebula rescues APP-induced STM
Impairments. (A) Aversive associative memory performance of 2-4 day-old flies 3
minutes after training (STM) for the indicated genotypes. n ≥ 18 independent trials per
genotype, * p < 0.05 compared to control, ** p < 0.05 between the indicated genotypes.

105

3.4.2 Nebula upregulation protects against APP-induced impairments in long-term
memory
We next examined whether Nebula upregulation also affected long-term memory
(LTM) defects by assaying the performance index 24 hours post a 10-trial spaced training
protocol (Tully et al. 1994). Fig 3.2 shows that APP upregulation caused severe defects
both immediately and 24 hours following training, indicating impaired memory
acquisition and retention. Despite an initial decrease in performance index immediately
following spaced training, flies overexpressing both APP and Nla performed almost as
well as control flies 24-hours following training, indicating impaired memory acquisition
and retention. These results indicate that Nla upregulation also effectively protects
against APP-induced LTM defects, but further suggests that repeated training may trigger
signaling pathways that feedback to affect the initial memory formation in APP and
Nebula overexpressing flies.

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Figure 3.2

Figure 3.2. APP-mediated impairments in long-term memory are rescued by
upregulating Nebula. (A) Quantification of memory performance immediately and 24-
hours after spaced training (LTM). n = 6 independent trials per genotype, ** p<0.05
comparing the indicated genotypes. Values indicate mean ± S.E.M.

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3.4.3 Nebula restores APP-mediated memory impairments through calcineurin
inhibition
Previous work has demonstrated that APP overexpression leads to calcineurin
hyperactivation and subsequent GSK-3β, but nebula overexpression inhibits both
perturbations through its ability to inhibit calcineurin (Shaw and Chang 2013). We
therefore test the hypothesis that aberrant calcineurin or GSK-3β signaling contributes to
APP-induced memory defects. To reduce calcineurin activation, we expressed UAS-
RNAi-calcineurin (RNAi-CaN) in APP overexpressing flies. Reduced calcineurin has
been shown to effectively restore calcineurin-mediated perturbations in axonal transport
in APP flies (Shaw and Chang 2013). Reducing calcineurin significantly improved STM
in flies with APP overexpression (APP;RNAi-CaN; Fig. 3.3.A; table S1), confirming that
abnormal calcineurin activity contributes to STM defects in this Drosophila model for
Alzheimer’s disease. On the other hand, reducing GSK-3β activation using a mutant of
fly GSK-3β (sgg in Drosophila) was not able to rescue APP-induced STM defects
(sgg
1
;APP; Fig 3.3.A). This suggests that activation of GSK-3β signaling is not the main
cause of memory defects in flies with APP overexpression.

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Figure 3.3

Figure 3.3 APP-mediated impairments in STM are rescued by restoring calcineurin
or PKA activity. (A) Short-term memory performance index for 2-4 day-old flies of the
indicated genotypes. n ≥ 18 independent trials per genotype, * p > 0.05 compared to
control, ** p < 0.05 between the indicated genotypes.

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Calcineurin has been shown to influence downstream cAMP-dependent protein
kinase (PKA) signaling, a kinase known for its importance in learning and memory
(Tully et al. 1994). We therefore tested the hypothesis that APP overexpression affects
short-term memory through PKA signaling. Fig. 3.3.A. shows APP-induced STM
significantly improved by wildtype PKA upregulation, supporting the hypothesis that
overexpression of APP also acts through PKA signaling to influence STM. To confirm
that upregulation of APP alters PKA signaling, we directly measured PKA enzymatic
activity using fly head extracts isolated form transgenic flies expressing the indicated
trasngene in all neurons. APP overexpression dramatically reduced PKA activity
compared to the control, whereas Nebula co-upregulation restored PKA activity close to
normal (Fig. 3.3.B). Consistent with previous reports, we find that manipulations of
Nebula alone also led to altered PKA activity (Fig. 3.3.B) (Chang and Min 2003). Note
that PKA upregulation did not perfectly rescue APP-induced STM defects, perhaps
because the exact level of PKA is crucial for normal memory. This is further supported
by data in which subtle changes in PKA signaling altered STM in mutants for PKA (dco)
as well as in the nebula mutant and overexpression models (Chang and Min 2003;
Yamazaki et al. 2007). Unfortunately, genetically increasing PKA using the UAS-PKA
line in the absence of APP failed to rescue memory impairments associated with aging
(S.3.4).
PKA is also a known regulator of LTM through its phosphorylation of CREB,
which is thought to activate CRE-dependent gene expression crucial for the establishment
of LTM (Yin et al. 1994; Drain et al. 1991) . Indeed, flies with APP overexpression
showed a correspondingly low level of CREB phosphorylation, whereas co-upregulation

110

of Nebula and APP restored CREB phosphorylation to normal (Fig. 3.3.C). These
findings imply that Nebula likely restored LTM defects by preserving CREB
phosphorylation.
Nebula is an important regulatory component of the CREB signaling pathway
important for long-term memory. Increased phosphorylation of CREB is mediated by
nebula’s capacity to inhibit calcineurin activity. Protein kinase A phosphorylates CREB
on Ser-133 to activate transcription in response to cAMP. cAMP binds to the regulatory
subunit of PKA and releases the active catalytic subunit from PKA. Previous research
confirms that the expression of RCAN1/DSCR1 increased the phosphorylation of CREB
and CRE-mediated gene transcription in response to the activation of the cAMP pathway.
We propose that nebula’s capacity to rescue APP-mediated impairments in learning and
memory is due to the negative regulation of the calcineruin signaling pathway and the
resulting effect of CREB phosphorylation.
Previous research in neuronal PC12 cells demonstrated that a mutant CREB in
which the Ser-133 residue of CREB was replaced by Alanine did not result in an increase
in CREB phosphorylation when RCAN1 was expressed (Kim et al. 2009). This result
indicates that increased phosphorylation of CREB by DSCR1 is specifically targeted to
the Ser-133 residue of CREB. In addition, it is likely that nebula’s ability to increase
cAMP-depent phosphorylation of CREB occurs in a PKA dependent manner. Previous
results suggest that co-expression of RCAN1 with the catalytic subunit of PKA caused
the enhanced phosphorylation of the Ser-133 residue of CREB. Inhibition of PKA with
the pharmacological inhibitor of PKA H-89 prevented the DSCR1-enhanced
phosphorylation of CREB (Kim et al. 2009). Similarly, treatment of neuron PC12 cells

111

with known calcineurin inhibitors, FK506 and cysclosporin A, resulted in a significant
accumulation of CREB phosphorylation indicating that calcineurin is a negative regulator
of CREB (Kim et al. 2009) and potential enhancer of long-term memory.

112

Figure 3.3 APP-mediated impairments in STM are rescued by Nebula’s capacity to
restore PKA and p-CREB. (B) PKA activity compared across genotypes. Quantification
represents assays normalized to the control. n = 12 independent experiments. (C) Western
blot analysis of phospho-CREB levels using nuclear extracts prepared form 2-4 day-old
flies. Co-upregulation of APP and Nebula restored phosphorylated CREB levels to that of
the control. The nuclear TATA binding protein (TBP) was used as a loading control. n =
8 independent experiments. All values are mean ± SEM. * p < 0.05 compared to to
control, ** p < 0.05 between the indicated genotypes.

113

3.4.4 Co-upregulation of APP and Nebula exacerbates short-term memory
impairments and exacerbates mitochondrial dysfunction in aging Drosophila
The finding that Nebula co-upregulation significantly improved the memory
performance of young flies with APP overexpression could potentially have important
clinical implications; we therefore assayed whether such protective effects persist into old
age. Similar to humans and other animal models, Drosophila show age-dependent
memory decline as demonstrated in the performance index for the control (Fig. 3.4.A)
(Yamazaki et al. 2007). However, despite restoring STM to normal in young flies less
than 10 days old, Nebula upregulation did not protect against APP-induced short-term
memory defects in older flies and instead further accelerated age-dependent memory
decline compared to the control (Fig. 3.4.A). This change is not due to altered
sensorimotor responses in older flies (table S.3.2). Together, these data reveal that nebula
upregulation initially acts to delay the onset of APP-induced memory loss, but
contributes to enhanced memory impairments in aged Drosophila. Interestingly,
employing RNAi strategy to reduce calcineurin protected against APP-mediated short-
term memory impairments in young Drosophila. Aged flies co-upregulating APP and
RNAi-CaN demonstrated age-dependent memory decline; however, the decline in
memory was less severe than co-upregulation of APP and nebula. The observation that
inhibiting calcineurin regulates memory differently than upregulating nebula suggests
that nebula has a secondary, calcineurin-independent effect on regulating memory
(Figure S.3.5).
What is the molecular or cellular switch underlying Nebula’s inability to protect
against APP-induced STM defects in aged flies? We first asked if Nebula is an

114

ineffective calcineurin inhibitor in older flies. The calcineurin activity assay revealed that
aging led to a significant increase in calcineurin activity even in the control flies by 45
days after eclosion (Figure 3.4.B). Nevertheless, Nebula’s upregulation still effectively
prevented APP-induced calcineurin hyperactivation compared to age-matched control
flies (Figure (3.4.B). Thus, change in Nebula’s ability to inhibit calcineurin is not a
primary cause of STM impairments in older flies with APP and nebula overexpression.
In addition to acting as an inhibitor of calcineurin, Nebula/DSCR1 has also been
shown to interact with the adenine nucleotide translocator (ANT) to regulate
mitochondrial function (Stoub et al. 2006; Rogalski and Mesulam 2009). We therefore
tested the hypothesis that nebula overexpression exacerbates mitochondrial dysfunction
during aging. First, we measured the cellular ATP content, which is indicative of the
overall mitochondrial function. Neuronal overexpression of APP or nebula alone in
young flies led to mitochondrial dysfunction as measured by the decrease in the overall
ATP content (Fig. 3.4.C). Interestingly, despite restoring APP-induced STM defects in
young flies, Nebula and APP co-upregulation still showed a similarly reduced ATP
content (Fig. 3.4.C), indicating that mitochondrial dysfunction, at least in young flies, is
not sufficient to trigger STM defects. In aged flies, however, Nebula and AP
overexpression significantly enhanced mitochondrial dysfunction by causing greater than
67% decrease in the overall ATP content (in a comparison of flies aged to 45 DAE vs. 3
DAE). The control and flies overexpressing APP only displayed about a 20% reduction in
the overall ATP content as a result of aging (Fig. 3.4.C). These data support the notion
that upregulation of Nebula during aging further exacerbates mitochondrial dysfunction.
Second, we measured the relative ROS levels in flies using dihydroethidium staining.

115

Chronic co-upregulation of APP and Nebula resulted in a significantly greater increase in
ROS levels during aging causes mitochondrial dysfunction and triggers ROS elevation
(Figure 3.4.D). Previous studies examining the link between oxidative stress and ROS on
STM revealed that STM of older flies is particularly sensitive to perturbation of genes
implicated in combating ROS, whereas young flies are not affected by decreases in
antioxidant enzymes or ROS elevation (Haddadi et al. 2014). Thus, the increase in ROS
and decline in ATP content seen in old flies overexpressing APP and Nebula likely
accelerated age-dependent memory decline. Reducing calcineurin by RNAi had the effect
of preventing a steep decline in ATP content in aged flies. In addition, Drosophila
expressing UAS-RNAi-CaN and UAS-APP did not have the same dramatic increase in
ROS observed in Droosphila upregulating UAS-Nebula
t1
. This finding suggests that in
addition to inhibiting calcineurin upregulation of Nebula influences other pathways that
impact ROS and ATP content.

116

Figure 3.4.A

Figure 3.4.A Co-upregulation of APP and Nebula enhances age-dependent memory
impairments and exacerbates mitochondrial dysfunctions. (A) Quantification of STM
during aging n ≥ 18 independent trials, * p < 0.05 compared to age-matched controls for
flies overexpressing APP or Nebula, * p<0.05 compared to age-matched control for flies
overexpressing APP, APP and Nebula, or Nebula alone.

117

Figure 3.4.B Co-upregulation of APP and Nebula enhances age-dependent memory
impairments and exacerbates mitochondrial dysfunctions. (B) Aging causes an
increase in calcineurin activity. Nebula still effectively inhibits calcineurin in 42-45 days
after eclosion (DAE) aged flies. N = 11 independent experiments.

Figure 3.4.C Co-upregulation of APP and Nebula enhances age-dependent memory
impairments and exacerbates mitochondrial dysfunctions. (C) Nebula upregulation
exacerbates decline in the overall ATP content in aged flies. All values were normalized

118

to young flies done in parallel experiments. N = 8 independent experiments for young
and old flies.

Figure 3.4.D Co-upregulation of APP and Nebula enhances age-dependent memory
impairments and exacerbates mitochondrial dysfunctions. (D) Pseudo-colored images
and quantification of dihydroethidium staining in the brains of young and aged
Drosophila. n ≥ 5 independent experiments per genotype per condition. All values
represent mean ± S.E.M. For (B), (C), and (D), * p<0.05 and † p<0.1 compared to age-
matched control; ** p < 0.05 between the indicated genotypes.

**
**
APP APP;nla
t1
nla
t1
+"
APP" APP;nla
t1"
Elav-GAL4
Elav-GAL4
*
*
2-day 45-day
Relative ROS
*
*
**
D
APP;
RNAi-Nla

APP;
RNAi-CaN

RNAiCaN

0"
0.5"
1"
1.5"
2"
2.5"
3"
Young"
Aged"
+
nla
t1"
APP;"
RNAi5Nla
"
APP;"
RNAi5CaN
"
RNAi5"
CaN
"
*
**"
**"

119

3.4.5 Acute Inhibition of Calcineurin rescues APP-mediated STM memory
impairments in aged flies

The bimodal effect of Nebula upregulation on APP-induced memory loss is age-
dependent, raising the possibility that either changes in basic cellular physiology during
aging affect Nebula’s ability to protect, or that acute vs. chronic upregulation of Nebula
exerts different effects on APP-induced memory loss. To explore these two possibilities,
we transiently upregulated APP with or without Nebula in either young or old flies using
an inducible mushroom body driver (MB-GeneSwitch-GAL4). MB-Gene-Switch-GAL4
allows temporal control of transgene expression specifically in the mushroom body
neurons following RU486 administration (Mao et al. 2004). We find that transient
upregulation of APP in the mushroom bodies of adult flies was sufficient to trigger STM
defects independent of sensorimotor defects (Fig. 3.5.A, Fig.3.5.B; table S.3.3).
However, acute co-upregulation of APP and Nebula in either young or aged flies
effectively rescued the APP-induced STM defect, indicating that aging alone is not the
cause of Nebula’s inability to rescue APP phenotypes. Furthermore, selective
upregulation of Nebula in old flies no longer impaired STM (unlike the young flies),
presumably by counter-balancing increased calcineurin activity in aged flies (Fig. 3.5.B).
If short-term instead of chronic upregulation of Nebula is indeed beneficial for
protecting against APP-induced memory loss, we reasoned that acute inhibition of
calcineurin should be able to ameliorate STM loss even in the presence of chronic APP
upregulation. To test this hypothesis, we pharmacologically blocked calcineurin by

120

feeding aged flies overexpressing APP with either 30 µM cyclosporine A or 30 µM
FK506, inhibitors of calcineurin. Excitingly, either cyclosporine A or FK506 effectively
rescued the STM defects seen in aged flies to values seen for age-matched control flies
(Fig. 3.5.C; table S.3.4). Acute inhibition of calcineurin also significantly improved the
STM of 45 day-old control flies, presumably because these drugs alleviated the increase
in calcineurin activity seen in aged control flies (Fig. 3.5.B and Fig.S.2.). Together, our
data suggest that Nebula’s inability to protect against memory loss in old flies with APP
overexpression is not due to irreversible damage caused by APP overexrpession; rather,
the memory impairment is likely due to chronic Nebula upregulation. These findings
further reveal that calcineurin dyshomeostasis contributes to age-dependent memory
impairment, and that acute inhibition of calcineurin can ameliorate memory deterioration
during aging in control and a fly model for AD. Nevertheless, we were not able to restore
STM to young control levels despite restoring calcineurin activity in old flies, suggesting
that aging may also trigger other cellular events that adversely affect memory
performance (Gupta et al 2013).

121

Figure 3.5 Acute inhibition of calcineurin by transient Nebula upregulation or
pharmacological treatment enhances STM in aged flies. (A) STM quantification for 2-
4 day-old flies and (B) 30-33 day-old flies with or without RU486 induction.
Performance was compared between uninduced and induced flies. ** p < 0.05. Transient
upregulation of APP severely impaired memory performance. Transient induction of both
APP and Nebula preserved memory performance in young and older flies. n = 6
independent experiments per genotype per condition in (A) and (B). (C) Acute
pharmacological inhibition of calcineurin reversed the STM defect to the level of age-
matched controls treated with vehicle control (DMSO). N = 5 independent experiments
per genotype. All values represent mean ± S.E.M. * p < 0.05 compared to vehicle feeding
of control flies; ** p < 0.05 comparing the indicated genotype.

122

3.5 Discussion
3.5.1 Summary
In Drosophila age dependent memory impairments are frequently associated with
changes in PKA signaling in the mushroom body. Previous work has found that large
decreases in PKA activity (~20% of wild-type) can inhibit memory (Drain et al. 1991).
PKA activity does not increase with aging leading to the conclusion that age- or APP-
dependent impairments in memory may be due to the accumulation of damage produced
by chronic PKA signaling or through acute and harmful changes during aging. The
differences in acute and chronic inhibition of calcineurin on memory performance have
not previously been examined in detail.
Previous studies demonstrated that Nebula/DSCR1 is upregulated in DS and AD
(Cook et al. 2005). Our findings reveal that Nebula/DSCR1 upregulation may contribute
to APP-induced memory impairments, but accelerating memory loss in aging. Our
finding that acute inhibition of calcineurin improved the memory performance of aged
control and APP overexpressing flies suggests the possibility of delaying and reversing
memory loss during aging. Our study therefore has wide implication for memory loss
during both natural aging and AD in DS. This research offers insight into mechanisms by
which restoring calcineurin or regulating Nebula/DSCR1 could function as a potential
therapeutic strategy for age dependent memory loss.

123

3.6 Supplementary Figures

Fig. S3.1 Memory deficits not attributed to differences in APP expression. (A)
Western blot depicting equivalent levels of amyloid precursor protein in the genotypes
with APP upregulation driven by the C739-GAL4, mushroom specific driver.
Quantification of N-APP levels were normalized to control and tubulin. n = 3
independent experiments.

124

Fig. S3.2 Treatment of Drosophila aged 42-45 days with CspA and FK506 effectively
inhibits calcineurin activity to levels of young controls. (A) Quantification of
calcineurin activity determined from extracts of old flies fed with either DMSO (vehicle)
as a control or drug. Drug-treated flies were treated for 3 days with either 30 µM CspA or
FK506. Quantification represents mean ± S.E.M. n = 5 independent experiments. ** p <
0.05 as compared to the indicated conditions.

125

Fig. S3.3 Age-dependent decline in STM performance in flies with altered PKA
activity. (A) Quantification of the performance index on a short-term memory task for
flies at 4 different points across aging: 2-4, 7-10, 30-33, and 42-45 DAE. PKA
upregulation alone partially rescues STM impairments across aging. Quantification
represents mean ± S.E.M. n = 12 independent experiments. *** p < 0.05 as compared
within genotypes.

0"
10"
20"
30"
40"
50"
60"
70"
Upregula)ng+PKA+alone+Par)ally+Rescues+STM+in+Aged+
Drosophila+
***
***
***
c739;+
c739/APP-PKA

C739/APP

Age (Day)
2
10 30 42

126

Figure S3.4. Reducing calcineurin in the presence of APP preserves STM
performance until 30-33 DAE when is stops protecting. (A) Quantification of the
performance index on a short-term memory task for flies at 4 different points across
aging: 2-4, 7-10, 30-33, and 42-45 DAE. Reducing calcineurin more effectively rescues
STM impairments in young flies when compared to increasing PKA activity.
Quantification represents mean ± S.E.M. n = 12 independent experiments. *** p < 0.05
as compared within genotypes.

c739;+
c739/APP
c739/APP-RNAi-CaN

C739;APP;PKA

0"
10"
20"
30"
40"
50"
60"
70"
Reducing"Calcineurin"Rescues"APP9Mediated"STM"Impairments"more"
effecCvely"than"Restoring"PKA"Levels"
Age (Day)
2 10 30 42
***
***
***
***

127

Figure S3.5. Human DSCR1 rescues STM performance for flies co-upregulating
APP and DSCR1. (A) Quantification of the performance index on a short-term memory
task for flies at 2-4 DAE confirming that human DSCR1 and fly nebula work in the same
pathway. Quantification represents mean ± S.E.M. n = 6 trials repeated over 3
independent experiments. *** p < 0.05 as compared within genotypes.

128

Figure S3.6. Reducing calcineurin with RNAi strategy does not exacerbate age-
dependent ATP decline to the same extent as upregulating nebula. (A) Quantification
of the ATP content for flies at 2-4 DAE and 42-45 DAE confirming that nebula
upregulation and calcineurin reduction function differently. Quantification represents
mean ± S.E.M. n = 5 independent experiments. *p < 0.05 as compared within genotypes.

0"
0.2"
0.4"
0.6"
0.8"
1"
1.2"
Control'
Elav/UAS/APP;RNAi/CaN'
Elav/UAS/RNAi/CaN'
Elav/'UAS/APP;Nebulat1'
Reducing'Calcineurin'with'RNAi'Prevents'the'
Age/Dependent'Decline'in'ATP'Associated'with'
Nebula'UpregulaBon'
1/3'DAE'
42/45'DAE'
***
***

129

Supplementary Table 1

Table S1. Sensorimotor responses of 2-4 day-old flies. Drosophila of all genotypes
avoided shock, MCH and Octanol. n = 6 independent trials per genotype and per
condition.

130

Table S2. Sensorimotor responses of 42-45 day-old flies. No change in sensorimotor
responses were observed across genotypes. n = 6 independent trials per genotype and
per condition.

131

Table S3. Sensorimotor responses of MB-GeneSwitch-GAL4 containing drivers
before and after induction. STM defects are not caused by changes in sensorimotor
responses. n = 6 independent trials per genotype and per condition.

132

Table S4. Sensorimotor responses of calcineurin inhibitor treated flies at 42-45-days
old are intact. n = 6 independent trials per genotype and per condition.

133

3.7 Acknowledgments
We thank members of the Chang and Sieburth laboratories for scientific
discussions. This work was supported by grants from the National Institute of Health
(NS080946), THE Jerome Lejeune Foundation, and Alzheimer’s Association and Global
Down Syndrome Foundation to KTC.

134

Chapter 4

4.1 Summary and Future Directions

Using the model organism D. melanogaster, I have demonstrated a role for
nebula/DSCR1 in ameliorating the impairments in axonal transport and memory caused
by upregulating amyloid precursor protein. Co-upregulation of APP and nebula in
neurons delays degeneration of photoreceptor axons and prevents accumulation of
membrane-bound organelles in the axons of larval nerves. This structural rescue
corresponded to a functional rescue as nebula upregulation restored locomotor behavior
in larvae and phototaxis in aging adult flies. Upregulation of nebula in the absence of
APP triggers impairments in short term-memory formation that is corrected by
upregulating nebula in the presence of APP – a surprising finding that suggests nebula’s
rescue is contingent on precise regulation of cell signaling. Nebula’s function in
regulating APP-mediated phenotypes is complicated by the observation that in young
flies nebula rescues memory impairments whereas in aging flies nebula exacerbates age-
dependent memory decline. This result indicates that nebula regulates a complex system
of signaling pathways that are downstream of calcineurin.
Downstream targets of calcineurin include glycogen synthase kinase 3 beta (GSK-
3β) and protein kinase A (PKA). Genetically manipulating expression of calcineurin,
GSK-3β, and PKA enabled an exploration of the role kinase and phosphatase activity
plays in regulating axonal transport and memory performance. I demonstrate that acute
inhibition of calcineurin may enhance short-term memory performance in normally aging
Drosophila and those upregulating APP in a diseased system.

135

4.2 Nebula Rescues APP-Mediated MBO Accumulation in the Axons
Nebula is highly conserved across species and its role as a calcineurin inhibitor
has previously been examined in the context of regulating reactive oxygen species,
mitochondria, and cell death. DSCR1 regulates the activation of the transcription factor
NFAT known to regulate immune function; however, for the scope of this research I
focused on neuronal inhibition of calcineurin the Ca
2+/
calmodulin-dependent
phosphatase. Using confocal microscopy, I show in Chapter 2 that upregulating APP in
the presence of constitutively active calcineurin exacerbates APP-axonal accumulation
phenotypes. In contrast, reducing calcineurin through nebula upregulation or reducing
calcineurin through RNAi eliminated this transport problem. This research offers the first
in vivo evidence for calcineurin signaling regulating axonal transport.
Another research team discovered that in the context of Huntington’s disease,
calcineurin inhibition through FK506 pharmacological administration led to sustained
phosphorylation of mutant huntingtin at S421 and restored its function in axonal transport
(Pineda et al. 2009). Calcineurin directly alters phosphorylation state on the huntingtin
protein leading to its negative regulation of neurotrophins such as brain-derived
neurotrophic factor. Calcineurin silencing in rat primary neuronal cultures expressing
mutant huntingtin resulted in restored anterograde and retrograde transport in neurons
(Pineda et al. 2009). This finding implicates increased calcineurin signaling in multiple
degenerative diseases and suggests that reducing calcineurin might effectively enhance
axonal transport in other disease models. I demonstrate in vivo that nebula restores
anterograde and retrograde transport of mitochondira and pre-synaptic vesicles.

136

Upregulation of nebula enhanced transport of APP-YFP in the anterograde direction.
Further investigation into whether nebula itself interacts with subunits of kinesin and
dynein motors could provide insight into how this protein enhances transport beyond its
regulation of cell signaling pathways necessary for cargo-motor interactions.

4.3 Nebula Rescues APP-Mediated Impairments in Cell Signaling
Upregulation of APP triggers an increase in calcium that activates the
phosphatase activity of calcineurin. Suprisingly, co-upregulation of nebula and APP also
led to an aberrant increase in calcium as reported by a genetically encoded calcium
sensor. This finding indicates that nebula’s protective mechanism of action is not through
correcting calcium influx; rather nebula restores calcineurin activity to levels observed in
the control overriding the APP-mediated increase in calcineurin. Drosophila co-
upregulating APP and constituitively active calcineurin had the highest levels of
calcneurin suggesting a link between altered phosphatase activity and severity of
aggregates. Calcineurin directly activates GSK-3β; however, increasing activity of GSK3
did not impair transport to the same severity as increasing calcineurin. GSK-3β has been
implicated in tau-associated pathologies of Alzheimer’s disease. Phosphorylation of Ser-9
of GSK-3β inhibits activity while phosphorylation at Tyr216 increases activity. I
demonstrate that nebula inactivates GSK-3 by enhancing phosphorylation at the Ser-9
site. In future work, it would be interesting to determine whether changes in GSK-3
activity negatively influences the hyperphosphorylation of tau and whether nebula can
intercept this pathology.

137

Impairments in axonal transport have been observed to have devastating
consequences in a variety of neurodegenerative diseases including Amyotrophic lateral
sclerosis, Huntington’s, Parkinson’s, and Alzheimer’s disease. Potential hypotheses
explaining the link between neurodegeneration and axonal transport defects include
deficiencies in delivering ATP-producing mitochondria to the synapse, protein
aggregation, deficits in the cargo’s ability to bind to the molecular motor, and the
inability of the molecular motor to bind to the microtubule. In this dissertation, I propose
that nebula through altering kinase and phosphatase can preserve synaptic function by
ensuring delivery of membrane-bound organelle to the terminal. Whether axonal
transport defects alone can cause impairments in cognitive function and learning and
memory in the absence of amyloid is an interesting future direction.
Glycogen synthase kinase is repeatedly implicated in exacerbating Alzheimer’s
pathology through its interaction with the microtubule-associated tau protein and
modulation of transcription factors that regulate apoptosis (Mines et al. 2011). Despite
the ability of GSK3-β to phosphorylate the kinesin light chain and negatively regulate
binding of cargoes to the motor, the role of this kinase in relationship to nebula had not
previously been unraveled. Reducing GSK-3β activity was more effective in preventing
the neurotoxicity attributed to Ab42 than reducing tau – a protein that when
hyperphosphorylated contributes to the neurofibrillary tangles associated with AD
neuropathology (Sofola et al. 2010; Mines et al. 2011). Phosphorylation state is critical
for GSK3-β activity. Calcineurin directly dephosphorylates GSK3 at the Ser-9 site.
Inhibiting calcineurin’s dephosphorylating activity increases the level of inactive GSK3-β
in comparison to total GSK3β. Nebula inhibits GSK3-β through the calcineurin pathway

138

resulting in enhanced axonal transport when both APP and nebula are expressed in
comparison to APP overexpression alone.

4.4 Two-Faced Regulation of Memory by Nebula
Understanding memory in normal individuals could provide insight into the
signaling pathways that are corrupted during neurodegenerative disease that robs them of
the memories that form their identity. All organisms undergo progressive age-dependent
memory decline. Questions remain as to how aging contributes to memory impairments.
In this dissertation, I demonstrate that acute inhibition of calcineurin in aged Drosophila
has the capacity to rescue age-dependent memory impairments. In future experiments, it
will be interesting to identify the genes that are upregulated and downregulated across
aging for Drosophila upregulating APP and those co-upregulating APP and nebula using
RNA-sequencing. The predominant theory for aging and memory decline is the free-
radical and oxidative damage model that suggests that reactive oxygen species cause
molecular damage to DNA that accumulate (Kirkwood and Austad, 2000). In support of
this theory, mutants with extended lifespans tend to have increased tolerance to free-
radicals and an increased oxidative stress response (Cysper and Johnson 1999). It will be
interesting to determine if nebula regulates genes responsible for responding to ROS.
Overexpression of APP has been found to significantly reduce the expression of
proteasome subunits (α-type 5 and β-type 7) that inhibit proteasomal degradation of
DSCR1 (Wu et al 2014). Recent research found that knockdown of DSCR1 reduced
APP-induced neuronal apoptosis. These data confirm the Jekyll and Hyde relationship of
DSCR1 in regulating APP-phenotypes. Over-expression of APP and DSCR1 may

139

contribute to disease pathogenesis in Alzheimer’s by enhancing neuronal apoptosis
through the proteasome pathway in later stages of disease progression despite initially
restoring memory through PKA signaling pathways.

4.4 Future Directions
Alzheimer’s disease is a debilitating, neurodegenerative disease wherein cell
death and synapse dysfunction in the hippocampus and frontal cortex destroy the
memories that form an individual’s identity. In the United States, epidemiological data
indicates that five million people are currently living with Alzheimer’s disease at an
estimated healthcare cost of 183 billion dollars annually. As the population ages, it is
believed that the prevalence will be 1 in every 45 Americans by 2040. Currently, there is
no known cause or cure for the decline in mental function that characterizes the disease
and leads to death.
By age 40, virtually all Down syndrome patients develop sufficient
neuropathology for a post-mortem diagnosis of Alzheimer’s disease; however, despite the
accumulation of amyloid-β plaques and neurofibrillary tangles, the severity of senile
plaques does not necessarily correlate with memory impairments, cognitive dysfunction,
and dementia. The percentage of DS adults with dementia jumps from 10% at age 40 to
25-65% by age 60 indicating that the onset of neurodegeneration occurs later than the
development of the pathology. Epidemiological data indicates that not all individuals
with Down Syndrome develop Alzheimer’s disease despite severe plaques and tangles;
therefore, it is important to evaluate the potential for transport defects and aberrant kinase
and phosphatase signaling to contribute more to AD than senile plaques and the

140

possibility that genes upregulated in DS play a neuroprotective role early on. The
observation that nebula/DSCR1 protects against APP-mediated axonal transport and
memory impairments in young flies but exacerbates ROS, mitochondrial dysfunction, and
memory deficits in aged flies supports the theory that DSCR1 upregulation plays a two-
faced role in regulating APP-induced phenotypes in Down syndrome patients.

141

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Abstract (if available)

Abstract The Down syndrome critical region 1 gene (also called regulator of calcineurin and Nebula) is located in the DSCR region of human chromosome 21 and upregulated in post-mortem brains from Down syndrome (DS) and Alzheimer’s disease (AD) patients. Questions remain as to whether elevated expression of DSCR1 is exacerbating disease pathology or delaying degeneration. The goal of this dissertation is to gain insight into the role of DSCR1/Nebula in Alzheimer’s disease. Over the course of my research, I explored the functional interaction between DSCR1/Nebula and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. I find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration, ameliorates axonal transport defects caused by APP overexpression, and preserves memory function in young flies by regulating phosphatase and kinase activity. ❧ APP upregulation results in the accumulation of pre-synaptic proteins in the axons and increases in calcineurin and glycogen synthase kinase 3 beta (GSK-3β) activity. Live-imaging experiments reveal that nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, these findings imply that correcting calcineurin and GSK-3β can prevent APP-induced pathologies. ❧ I further investigate the effects of DSCR1/Nebula upregulation on amyloid precursor protein-induced learning and memory deficits. I find that while upregulation of nebula alone impairs memory, co-upregulation of APP and nebula can effectively restore APP-induced memory defects, but only in young Drosophila. Nebula/DSCR1 overexpression rescues perturbations in calcineurin and cAMP signaling caused by APP overexpression. Surprisingly, nebula accelerated age-dependent memory impairments, increased reactive oxygen species, and enhanced mitochondrial dysfunction in aged flies. This finding suggests that upregulation of DSCR1 may delay APP-induced memory problems initially but enhances memory decline at a later age. My data further demonstrates that acute upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation

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Asset Metadata

Creator Shaw, Jillian Lee Satter (author)

Core Title Nebula/DSCR1 upregulation preserves axonal transport and memory function in a Drosophila model for Alzheimer's disease

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 03/12/2015

Defense Date 12/11/2014

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

Tag Alzheimer's disease,axonal transport,calcineurin,Down syndrome,Drosophila,DSCR1,GSK3,memory,Nebula,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chang, Karen T. (committee chair), Chen, Jeannie (committee member), Jakowec, Michael (committee member), Miller, Carol Ann (committee member), Sieburth, Derek (committee member)

Creator Email jillials@usc.edu,Jillianlshaw@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-538930

Unique identifier UC11297290

Identifier etd-ShawJillia-3230.pdf (filename),usctheses-c3-538930 (legacy record id)

Legacy Identifier etd-ShawJillia-3230.pdf

Dmrecord 538930

Document Type Dissertation

Format application/pdf (imt)

Rights Shaw, Jillian Lee Satter

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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