A Thesis

entitled

Role of DSCAM in Netrin-1 Mediated

Axon Repulsion and Neuronal Migration

by

Anish A. Purohit

Submitted to the Graduate Faculty as partial fulfillment of the

requirements for the Master of Science in Biology

______Dr. Guofa Liu, Committee Chair

______Dr. Richard Komuniecki, Committee Member

______Dr. Scott Leisner, Committee Member

______Dr. Malathi Krishnamurthy, Committee Member

______Dr. Robert Steven, Committee Member

______Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo August 2011

Copyright 2011, Anish A. Purohit This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Role of DSCAM in Netrin-1 Mediated Repulsion and Neuronal Migration by

Anish A. Purohit

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Biology

The University of Toledo August 2011

Axon pathfinding and neuronal migration are key events for proper nervous system development and function. Neuronal growth cones explore the environment for extracellular cues, which guide the along specific paths towards their targets.

Netrin-1, a bifunctional guidance cue, binds to receptors deleted in colorectal cancer

(DCC) and Down syndrome cell adhesion molecule (DSCAM) mediating axon attraction, and uncoordinated-5 (UNC5) mediating axon repulsion. The DCC/UNC5 heterodimer also modulates netrin-1 repulsion in the developing nervous system. We found that

DSCAM interacts with UNC5C and netrin-1 stimulation increases this interaction in postnatal cerebellar cells. Therefore, we hypothesize that DSCAM may play a role in netrin-1 repulsion. Here we show that in an in vitro collapse assay, netrin-1 induces mouse cerebellum external granule layer (EGL) cell growth cone collapse. The

DSCAM intracellular domain modulates netrin-1 induced axon repulsion. Inhibiting

DSCAM intracellular signaling causes aberrant neuronal migration in the postnatal cerebellar EGL cells ex vivo. Thus, DSCAM plays a critical role in netrin-1 repulsion.

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To My Family and Kinjal…

Who have made Life worth living for…

Contents

Abstract ...... iii Contents ...... v List of Figures ...... i vii List of Abbreviations……………………………………………………………………………... x

1 Background ...... 1 1.1 The Nervous System ...... 1 1.2 ...... 1 1.3 Discovery of the First Guidance Cue – ephrin-As ...... 3 1.4 Netrins ...... 3 1.4.1 Structure ...... 3 1.4.2 Netrin Function ...... 4 1.5 Deleted in Colorectal Cancer (DCC) ...... 5 1.5.1 Protein Structure ...... 5 1.5.2 DCC Function ...... 5 1.6 Uncoordinated-5 (UNC5) ...... 6 1.6.1 Protein Structure ...... 6 1.6.2 UNC5 Function...... 7 1.6.3 The Developing Mouse Cerebellum ...... 7 1.6.4 Netrin-1 repels UNC5C expressing granule cells ...... 9 1.6.5 UNC5C/DCC mediating netrin-1 repulsion ...... 10 1.7 Down syndrome cell adhesion molecule (DSCAM) ...... 10 1.7.1 Protein Structure ...... 11 1.7.2 DSCAM Function ...... 11 2 Significance...... 12 v

3 Materials and Methods ...... 14 3.1 Plasmids and Contructs ...... 14 3.2 ...... 15 3.3 Immunoprecipitation and Western Blot Analysis ...... 15 3.4 Dissociated Primary Neuron Culture ...... 16 3.5 Growth Cone Collapse ...... 16 3.6 Ex vivo Cerebellar Slice Electroporation and Culture ...... 17 3.7 Cryosection...... 18 3.8 Immunostaining ...... 18 4 Results ...... 20 4.1 Biochemical Analysis of DSCAM and UNC5C Interaction ...... 20 4.1.1 Interaction of DSCAM and UNC5C in transfected HEK 293 cells ...... 20 4.1.2 Interaction of endogenous DSCAM and UNC5C in E15 cortical neurons .. 21 4.1.3 Time course of the netrin-1-induced endogenous DSCAM and UNC5C interaction in E15 cortical cells ...... 22 4.1.4 Time-dependent induction of endogenous DSCAM and UNC5C interaction in P2 cerebellar neurons by netrin-1 ...... 23 4.1.5 The Domain Interaction of DSCAM and UNC5C in HEK 293 cells ...... 24 4.1.6 RNAi knockdown of UNC5C in transfected HEK 293 cells ...... 25 4.2 The DSCAM/UNC5C expression pattern in embryonic and postnatal mouse dissociated neurons and brain slices...... 26 4.2.1 Co-expression of DSCAM and UNC5C in dissociated E15 mouse cortical and cerebellar neurons, and P2 cerebellar neurons...... 27 4.2.2 The expression pattern of DSCAM and UNC5C in the P4 cerebellar and cortical slices ...... 28 4.3 Netrin-1 induced EGL cell growth cone collapse ...... 30 4.3.1 The role of DSCAM in EGL cell growth cone collapse ...... 30 4.3.2 The role of UNC5C in the EGL growth cone collapse ...... 32 4.3.3 The combined role of DSCAM and UNC5C in netrin-induced growth cone collapse ...... 34 4.4 The ex vivo cerebellar slice electroporation and culture assay...... 37 5 Discussion ...... 42 vi

6 References ...... 51

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List of Figures

4-1 DSCAM and UNC5C interaction in transfected HEK 293 cells. 20

4-2 DSCAM and UNC5C interacted endogenously in E15 mouse cortical neurons. 21

4-3 The interaction of endogenous DSCAM and UNC5C increased after netrin-1 stimulation in E15 mouse cortical cells. 22

4-4 DSCAM and UNC5C interaction increased over time with netrin-1 stimulation in P2 cerebellar neurons. 23

4-5 The DSCAM extracellular domain interacted with UNC5C. 24

4-6 The UNC5C Ig2 domain interacted with DSCAM. 25

4-7 RNAi knockdown of UNC5C in transfected HEK 293 cells 26

4-8 DSCAM and UNC5C are coexpressed in dissociated E15 mouse cortical and cerebellar, and P2 cerebellar neurons. 27

4-9 The co-expression of DSCAM and UNC5C in P4 cortical and cerebellar slices. 28

4-10 DSCAM was required for netrin-1 induced EGL cell growth cone collapse. 30

4-11 DSCAM mediated netrin-1 repulsion in EGL cells. 31

4-12 UNC5C mediated netrin-1 induced EGL cell growth cone collapse. 32

4-13 UNC5C mediated netrin-1 repulsion in EGL cells. 33

4-14 Simultaneous knockdown of DSCAM and UNC5C abolished netrin-1 induced EGL cell growth cone collapse. 34

4-15 Simultaneous knockdown of DSCAM and UNC5C blocked netrin-1 induced growth cone collapse in EGL cells. 35 viii

4-16 DSCAM knockdown increased EGL cell migration rate after 2 DIV. 37

4-17 UNC5C knockdown slowed migration rate while both DSCAM and UNC5C knockdown increased neuronal migration rate. 38

4-18 Knockdown of DCAM and UNC5C did not change migration rate significantly in EGL neurons after 1 day in culture. 39

4-19 Knockdown of DSCAM and UNC5C changed migration rate in EGL neurons after 2 days in culture. 40

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List of Abbreviations

C. elegans…………………………...Caenorbabditis elegans cAMP………………………………. Adenosine-3´,5´-cyclic monophosphate cGMP………………………………. Guanosine-3´,5´-cyclic monophosphate DBD………………………………... DCC binding domain DCC………………………………... Deleted in colorectal cancer DD………………………………….. Death domain DSCAM……………………………. Down syndrome cell adhesion molecule E……………………………………. Embryonic EGF………………………………… Epidermal EGL………………………………… External granule layer FAK………………………………... Focal adhesion kinase FNIII……………………………….. III repeats GC………………………………….. Granule cell GPI…………………………………. Glycophosphatidylinositol HEK………………………………... embryonic kidney Ig…………………………………… Immunoglobulin IGL…………………………………. Inner granule layer lRL…………………………………. Lower rhombic lip ML…………………………………. Molecular Layer MLB………………………………... Mild lysis buffer P……………………………………. Postnatal PCL………………………………… Purkinje cell layer PLL………………………………… Poly-L-lysine r1…………………………………… Rhombomere 1 RGC………………………………... Retinal ganglion cell Tsp…………………………………. Thrombospondin UNC5………………………………. Uncoordinated-5 UNC-6……………………………… Uncoordinated-6 uRL………………………………… Upper rhombic lip WM………………………………… White matter WT…………………………………. Wild type

x

Chapter 1

Background

1.1 The Nervous System

In most if not all organisms, the nervous system is a group of neuronal circuits or networks specialized to process incoming information and outputting responses. The vertebrate nervous system is divided into two parts, the central (CNS) and the peripheral

(PNS) nervous system. The CNS consists of the brain and the , acting as the command center for processing information and creating appropriate responses, while the

PNS receives information from the environment and conducts CNS outputs.

Neuron is the basic functional unit in the nervous system. The neuronal structure is divided into three main parts: dendrites, soma or cell body, and the axon. Dendrites transmit incoming signals from other neurons or cells to the soma. The cell body receives, processes, and propagates this information to the axon via electrochemical signals. The axon protrudes from the cell body and carries the signal to other neurons or cells via .

1.2 Axon Guidance

Axon pathfinding and neuronal migration are key events for proper nervous system development and function. In the developing nervous system, neurons migrate to

1 their final destinations and axons have to project to their final targets (Guan and Rao,

2003). Neurons or axons may migrate far distances before reaching their final targets.

Both developmental events require guidance cues in the extracellular environment (Zou and Lyuksyutova, 2007). Guidance cues are like traffic signs, guiding or stopping neurons and axons entering certain areas during their trip. The four major canonical guidance cues are netrins (Colamarino and Tessier-Lavigne, 1995; Kennedy, 2000; Merz and Culotti, 2000), semaphorins (Kolodkin and Ginty, 1997; Raper, 2000), ephrins

(Flanagan and Vanderhaeghen, 1998; O’Leary and Wilkinson, 1999), and Slits (Wong et al., 2002; Wu et al., 1999). Except for ephrin-A family and a subset of semaphorins located on the plasma membrane, guidance cues are secreted defining a strict gradient in the extracellular environment (Guan and Rao, 2003). Guidance cues can either attract or repel navigating axons or neurons. The netrins, ephrins, and semaphorins can act as bifunctional molecules, attracting or repelling axons. Slits have been shown to be repellents for different axon projections (Huber et al, 2003). These extracellular guidance cues lay out a specific grid for axons and neurons to navigate in the developing nervous system. How does a growth cone choose among a plethora of guidance mechanisms? The growth cone displays specific guidance receptors on the cell membrane. The interaction between guidance cues and receptors initiates specific downstream signaling cascades that will ultimately steer the growth cone towards the proper direction (Huber et al.,

2003). For example, if a growth cone senses a strong gradient, intracellular repulsion signaling events will guide the axon in a direction away from the Slit gradient source. But in the developing environment, axons have to navigate through overlapping gradients,

2 increasing the complexity and crosstalk between intracellular signaling cascades (Huber et al., 2003).

1.3 Discovery of the First Guidance Cue – ephrin-As

The first guidance cue was discovered during the mid to late 1900s. Initially,

Roger Sperry observed retinal ganglion cells (RGCs) projecting axons in a specific pattern towards the optic tectum in frogs and chickens. The outer layer of the retina contains RGCs that project axons underlying the vitreal surface that travel towards the optic disk. The bundle of axons later becomes the optic nerve. In frogs, the axons all cross the towards the optic tectum. If the retina was split into four quadrants

– temporal, nasal, rostral (up), and caudal (down) – each set of axons for each retina will cross the chiasm and project to different parts of the tectum. Sperry originally hypothesized that due to the high specificity of axon projections towards targets, axons have a chemoaffinity for such cues. Later, the guidance cue was purified from chicken tectum leading to the discovery of ephrin-As as the guidance cue in the tectum mediating repulsion through Eph receptors expressed on RGCs (Sperry, 1963).

1.4 Netrins

1.4.1 Protein Structure

Netrins are a set of bifunctional guidance cues conserved across many species, ranging from (C. elegans) and , to vertebrates (Ishii et al., 1992; Bernhardt et al., 1992; Kennedy et al., 1994). Originally, genetic screens in C.

3 elegans led to the discovery of Uncoordinated-6 (UNC-6) in axon guidance (Hedgecock et al., 1985; Hedgecock et al., 1987). UNC-6 was found to have similar protein to (Ishii et al., 1992). UNC-6 became known as netrin, after purification from embryonic chicken brains, when the floor plate was discovered to enhance axon outgrowth and attract commissural axons within the mouse spinal cord

(Serafini et al., 1994; Tessier-Lavigne et al., 1988).

In vertebrates, netrins 1-4 are secreted proteins of the family, compared to netrin G1 and G2 that are glycophosphatidylinositol (GPI)-linked plasma membrane proteins. The N-terminal portion of netrins, similar to laminins, contain a globular domain VI and three epidermal growth factor (EGF) repeat domain V. Netrin N-terminal domains V1-3 are similar to laminin γ subunits while netrin-4 and –Gs are similar to β laminin subunits. Domain VI and V bind to Deleted in Colorectal Cancer (DCC) and the

Uncoordinated-5 (UNC5) family receptors, respectively (Rajasekharan and Kennedy,

2009). In C. elegans, UNC-6 domain VI is important for axon attraction and repulsion, while V2 or V3 is involved in repulsion (Wadsworth et al., 1996; Lim et al., 2002). C domain does not play a role in axon guidance (Wang et al., 2002). All netrins are expressed in mammals, except netrin-2 is only expressed in chicken and

(Moore et al., 2007).

1.4.2 Netrin Function

Netrins have the ability to attract or repel axons by activation of two canonical receptor families: 1) DCC (C. elegans UNC-40 and Drosophila Frazzled); 2) UNC5 (C. elegans UNC-5 and Drosophila Dunc5). In C. elegans, UNC-6 is necessary to attract ventrally migrating pioneer axons through UNC-40 and dorsally migrating axons through

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UNC-5. In vertebrates, netrin-1 function was well studied in the developing spinal cord, where dorsally derived commissural neurons projected axons towards the ventral floor plate (Kennedy et al., 1994; Serafini et al., 1996). Netrin-1 knockout mice exhibit many phenotypic defects in axon guidance, including a reduction in the number of commissural axons, shortening and misrouting of axons in the developing spinal cord, lack of the and hippocampal commissure, reduction in the size of the anterior commissure and absence of the pontine nuclei in the brain stem (Serafini et al., 1996).

1.5 Deleted in Colorectal Cancer (DCC)

1.5.1 Protein Structure

The vertebrate DCC protein contains four immunoglobulin (Ig) domains and six fibronectin III (FNIII) repeats within the extracellular domain and three large cytoplasmic motifs termed P1-3 (Huber et al., 2003). The FNIII repeats are involved in netrin-1 binding (Bennett et al., 1997; Geisbrecht et al., 2003; Kruger et al., 2004). In the cytoplasmic domain, P1-3 regions contain protein binding and phosphorylation sites. The

P2 domain is proline-rich containing four SH3 motifs and the P3 domain contains possible phosphorylation sites.

DCC can homodimerize, upon netrin-1 stimulation, through P3-P3 domain binding resulting in attraction (Stein and Tessier-Lavigne, 2003).

1.5.2 DCC Function

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In the developing vertebrate spinal cord, dorsal commissural axons project ventrally towards the floor plate. DCC is expressed on the surface of commissural axons, mediating netrin-1 attraction (Fazeli et al., 1997). Both DCC and netrin-1 knockout mice show similar phenotypes in the developing nervous system (Fazeli et al., 1997; Serafini et al., 1997).

After netrin-1 binds to DCC, how does the growth cone trigger downstream signaling to guide the axon turning towards the attractant? Previous studies have shown that many signal molecules are involved in netrin/DCC signaling, such as adenosine-

3´,5´-cyclic monophosphate (cAMP)/guanosine-3´,5´-cyclic monophosphate (cGMP), the

Rho family of small GTPases, Rac and Cdc42, Nck1, focal adhesion kinase (FAK), Fyn,

Pak, Src, Ena/Vasp, DOCK180 and N-WASP (Shekarabi et al., 2005; Li et al., 2002; Li et al., 2004; Meriane et al., 2004; Lebrand et al., 2004, Li et al., 2004; Li et al., 2006; Liu et al., 2004 Luo, 2000; Dickson, 2001; Shekarabi et al., 2005; Causeret et al., 2004).

These proteins are required for netrin-1-induced axon outgrowth and attraction.

Phosphoinositide breakdown, by phosphatidylinositol-3-kinase, through activation, releases intracellular calcium necessary for growth cone turning (Ming et al.,

1999; Rhee, 2001; Hong et al., 2000).

1.6 Uncoordinated-5 (UNC5)

1.6.1 Protein Structure

There are four vertebrate UNC5 homologs, UNC5A-D or UNC51-4, opposed to just one homolog in C. elegans and Drosophila. The UNC5 extracellular domain contains two Ig domains and two thrombospondin (Tsp) domains, and its intracellular domain

6 contains a ZU-5, DCC binding domain (DBD), and death domain (DD). The protein sequence of the ZU-5 domain is similar to the Zona Occludens-1 scaffolding protein found in tight junctions (Itoh et al., 1997). In C. elegans, the cytoplasmic region between

ZU-5 and DD is crucial in the downstream protein binding. The DCC binding domain was revealed and shown to function in netrin-1 long-range repulsion, while UNC5 alone can function in short-range netrin-1 repulsion (Keleman and Dickson, 2001; Hong et al.,

1999; Killeen et al., 2002). (See section UNC5/DCC mediating netrin-1 repulsion).

1.6.2 UNC5 Function

Trochlear motor axons expressing UNC5 steered away from netrin-1 in the developing spinal column (Figure 8) (Colamarino et al., 1995). UNC5 can mediate short- range netrin-1 repulsion in Drosophila (Keleman and Dickson, 2001). Previous studies have shown that UNC5 is involved in netrin-1 mediated repulsion in the developing cerebellum.

1.6.3 The Developing Mouse Cerebellum

The postnatal mouse cerebellum system, like the cortex, is a great model for studying neuronal migration due to the development of cell-specific layers.

The initial development of the mouse cerebellum arises from cells fated by specific factors secreted by the isthmus organizer at the midbrain-hindbrain boundary.

The organizing center mediates the development of the mesencephalon and the metencephalon. A constriction forms from the caudal mesencephalon and the rostral metencephalon – the future anterior boundary of the cerebellum. At the caudal portion of the metencephalon, a constriction segments the hindbrain into rhombomeres 1 and 2 (r1 and 2). The r1 gives rise to the rhombic lip, a structure formed between the dorsal

7 neuroepithelium and the expanding fourth ventricle roof plate (the source of netrin-1)

(Donkelaar et al., 2003). By embryonic (E) 9.5, the rhombic lip is defined at the edge of the ventricle roof plate (the r1 region or the upper rhombic lip) and the hindbrain boundary (the lower rhombic lip). The upper rhombic lip (uRL) will generate immature migrating neuronal progenitors (migration begins between E9.5 and E11.5), eventually creating the cerebellar primordium and lateral pons (Przyborski et al., 1998). The lower rhombic lip (lRL) will generate the pontine and reticulotegmental nuclei and the inferior olive (Gilthorpe et al., 2003).

From E13, the uRL contains mitotic cells, along the dorsolateral alar plate, migrating in a rostromedial direction to form the external granule layer (EGL). The EGL cells migrate away from the netrin-1 source in the fourth ventricle. From E13.5 to E17.5 mitotically active EGL cells migrate inward into the cerebellum primordium to create the cerebellum. Grooves are created at the cerebellum surface, which eventually create distinct fissures throughout the cerebellum (Przyborski et al., 1998).

For the first two postnatal weeks, EGL cells undergo mitosis about once a day and stop for about two days, all while extending two short processes horizontally and migrating tangentially to the deeper EGL. Rate of migration slows and processes elongate during the middle to inner EGL. Once in the molecular layer (ML), Granule cells (GCs) switch from the tangential to radial migration. A third process extends vertically downward via attachment to Bergmann glial fibers. GCs reach the Purkinje cell layer

(PCL) and detach from glial fibers. GCs round up and halt migration during a brief stationary phase. The somas and vertical processes elongate again and migrate into the inner IGL. As the vertical process reaches the white matter (WM), GC migration arrests

8 near the IGL-WM border. The two horizontal processes in the ML become parallel fibers synapsed with Purkinje cells dendrites (Donkelaar et al., 2003).

1.6.4 Netrin-1 repels UNC5C expressing granule cells

The uRL cells start to migrate over the anlage around E9.5 due to the strong netrin-1 source at the basal plate of the fourth ventricle and along the outer mesencephalon (Przyborski et al., 1998). Hence, the migrating EGL cells are repelled by netrin-1. Postnatal EGL explant axons are repelled by netrin-1 in the co-culture assay

(Alcantara et al., 2000).

Interestingly, EGL cells were seen migrating out of the explants away from netrin-1 aggregates (Alcantara et al., 2000). UNC5C and netrin-1 mRNAs are highly expressed in granule cells migrating from the external (EGL) to inner granule layer (IGL) in the developing cerebellum (Alcantara et al., 2000).

Therefore, UNC5C is believed to canonically mediate netri-1 repulsion in the developing cerebellum. UNC5C deficient mice demonstrated aberrant migration phenotypes during the cerebellar development.

Clearly, many granule cells have lost sensitivity to netrin-1 and cannot be repelled in the proper direction. Many granule cells are seen migrating into the midbrain region

(towards the netrin-1 source that creates the midbrain/cerebellum boundary). Also, granule cells have created an enlarged WM. When vertical projecting EGL axons contact the IGL/WM boundary, axon outgrowth is halted and the soma will retract to the bottom of the IGL. Netrin-1 expressed in the developing fourth ventricle can possibly repel GCs from migrating aberrantly into the WM by the long-range repulsion. How can netrin-1 exert a long-range repulsive effect on growing axons and migrating neurons?

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1.6.5 UNC5C/DCC mediating netrin-1 repulsion

While UNC5 alone can mediate netrin-1 induced axon repulsion over short range, when bound to DCC, dependent on netrin-1, UNC5 can mediate the long range repulsion

(Kaleman and Dickson, 2001).Interestingly, an in vitro growth cone steering assay converted spinal cord commissural axon attraction by netrin-1 into repulsion after UNC5B transfection. The binding of DCC and UNC5 cytoplasmic domains is netrin-1 dependent and UNC5 cytoplasmic domain is only necessary to convert DCC attraction into netrin-1 induced long-range repulsion (Hong et al., 1999).

However, many UNC5C deficient cells still migrate away from netrin-1 in the developing cerebellum. Could other receptors mediate netrin-1 repulsion? Many rostral cerebellar cells lack UNC5C, which questions their ability to migrate ectopically from netirn-1 source as well (Ackerman et al, 1997).

1.7 Down syndrome cell adhesion molecule (DSCAM)

Down syndrome (DS) causes mental retardation with defined brain abnormalities.

Defects occur in the cortex, hippocampus, and cerebellum. DS patients have mutations in a specific region mapped to 21, the same region encoding

(Yamakawa et al., 1998). DS patients have a smaller cortex and cerebellum, possibly resulting from DSCAM overexpression (Jernigan and Bellugi, 1990; Jernigan et al.,

1993). DS patients and Ts65Dn mice (overexpression of DSCAM) show decreased

Purkinje cell numbers in the Purkinje cell layer (PCL) and granule cells in the EGL and

IGL. DSCAM is highly expressed in the PCL, with lower levels in the EGL and IGL.

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1.7.1 Protein Structure

DSCAM is a transmembrane protein belonging to the immunoglobulin (Ig) superfamily. The extracellular domain of DSCAM contains ten Ig domains and six fibronectin III (FNIII) repeats (Zhu et al., 2011).

1.7.2 DSCAM Function

Drosophila has four dscam : dscam1-4. dscam1 alternative splicing can potentially generate about 38,000 different Dscam proteins (Schmucker et al., 2000) and these isoforms interact through homophilic binding (Wojtowicz et al., 2004). In vertebrates, DSCAM is involved in neuronal self-avoidance for dendrite arborization

(Soba et al., 2007; Fuerst et al., 2008). In DSCAM deficient mice, retinal ganglion cells

(RGCs) have defects in neuronal spacing and dendritic arborization patterns (Fuerst et al,

2009). Studies in the chicken retina have shown that DSCAM plays a role in formation (Yamagata et al., 2008). These results suggest that DSCAM can function in different pathways.

In DCC knockout mice, a majority of commissural axons in the developing spinal cords are misguided, yet some axons still reach the floor plate, suggesting another guidance mechanism plays a role in guiding commissural axons (Fazeli, et al., 1997).

Recent studies have shown that DSCAM functions as a new netrin-1 receptor mediating commissural axon attraction (Andrews et al., 2008; Liu et al., 2009; Ly et al., 2008).

DSCAM and DCC may collaborate in netrin-1 attraction; however, whether

DSCAM directly interacts with DCC is still under debate. In Drosophila, Dock and Pak are involved in netrin-1/DSCAM signaling (Schmucker et al., 2000).

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Chapter 2

Significance

In the developing cerebellum, UNC5C deficient granule cells migrate ectopically into the midbrain regions and the cerebellar white matter (WM)

(Przyborski et al., 1998; Alcantara et al., 2000). Ectopic migration into the midbrain regions can result from functional loss of netrin-1 repulsion. EGL cells migrate into the WM probably due to loss of repulsion from the netrin-1 source located in the fourth ventricle zone, suggesting netrin-1/UNC5C long-range repulsion is lost.

However, UNC5C aberrant granule cell migration phenotypes cannot be explained alone by a netrin-1/UNC5C mechanism. Netrin-1 knockout mice show very few granule cell ectopias in the white matter and midbrain regions, suggesting another mechanism also facilitates in cerebellar granule cell migration.

Current studies indicate that DSCAM is involved in mediating netrin-1 attraction.

However, the mRNA expression analysis shows DSCAM expression in the developing cerebellum, a region where netrin-1 repulsion dominates. This mRNA expression of

DSCAM coincides with that of UNC5C and netrin-1 in the EGL. Why is DSCAM expressed in the developing cerebellum, where canonically netrin-1/UNC5C mediate repulsion? Does DSCAM also play a role in a netrin-1 repulsion system? Genetic and medical evidence also provide clues for DSCAM function in the cerebellum. dscam

12 lies within the extra copy of chromosome 21 region associated with Down syndrome

(Yamakawa et al., 1998). Down syndrome patients have a smaller cerebellum, possibly leading to cognitive defects associated with improper wiring of the cerebellum circuitry. These phenotypes could be a possible result of overexpression of DSCAM.

Also, UNC5 can bind to DCC to switch DCC attraction into repulsion, raising the possibility for other guidance receptors to interact with UNC5. DCC binds to netrin-1 but the repulsive signal is mediated by the UNC5 intracellular domain. The protein structure of DSCAM is similar to DCC and both receptors are required for netrin signaling.

Thus, DSCAM may play a role in netrin-1 repulsion by interacting with UNC5C.

We hypothesize that: 1) DSCAM and UNC5C interact, 2) DSCAM plays a functional role in netrin-1 repulsion in the developing cerebellum. This study will shed light on a new mechanism for netrin-1 signaling in the developing nervous system.

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Chapter 3

Materials and Methods

3.1 Plasmids and Contructs

For biochemical analysis in HEK 293 cells, the following constructs were used:

DSCAM-Flag, DSCAM∆C-Flag (human DSCAM without the intracellular domain),

DSCAM∆N-Flag (human DSCAM without the extracellular domain) UNC5C-HA,

UNC5C∆Ig1-HA, UNC5C∆Ig2-HA, UNC5C∆Igs-HA, and UNC5C∆Tsps-HA (Kruger et al., 2004).

For nucleofection and electroporation, constructs used were: Venus-YFP,

DSCAM-Flag, DSCAM∆C-Flag, DSCAM∆N-Flag, mU6 (empty shRNA vector),

DSCAM shRNA1, DSCAM shRNA2 (control), UNC5C-HA, UNC5C∆C-HA,

UNC5C∆N-HA, UNC5C shRNA5, and UNC5C shRNA6 (control). The targeted sequences of DSCAM shRNA1, DSCAM shRNA2, UNC5C shRNA5 and UNC5C shRNA 6 are: AAAGAGTTTAGCTGAAATGCT, AATGCATCTCTGCAAGAGGTA,

AAGAACCCAAGGCTCTTCATT and AACTGTACTGTGTCAGAGGAA.

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3.2 Antibodies

Western Blot

Primary Antibodies: anti-Flag 1:5000 (Abcam), anti-HA 1:1000 (Santa Cruz), anti-DSCAM 1:2000, and anti-UNC5C 1:500 (R&D).

Secondary Antibodies: anti-Rabbit 1:10,000 (Santa Cruz) and anti-Goat 1:5,000 (Santa

Cruz).

Immunohistochemistry and Immunocytochemistry

Primary Antibodies: anti-DSCAM 1:1000, anti-UNC5C 1:20, anti-Calbindin

1:300 (Sigma), Phalloidin 1:40 (Invitrogen) and DAPI 1:1000.

Secondary Antibodies: anti-Rabbit Cy2, anti-Goat Cy3, anti-Mouse Cy3.

3.3 Immunoprecipitation and Western Blot Analysis

HEK cells were transfected with 5 μg Unc5C-HA and DSCAM-Flag using transfection method PEI at 2:1 ratio with DNA. 4x106 dissociated E15 cortical and P4 cerebellar cells were plated on Poly-L-lysine (PLL) -coated 35 mm tissue culture dishes.

After 24 hours, cells were starved in starvation media (DMEM + 0.1% Bovine Serum

Albumin (BSA) + 1X Penicillin/Streptomycin) for at least 6 hours to overnight for HEK

293 cells or 4 hours for neurons. Cells were stimulated for 20 mins with HEK or netrin-1 conditioned media. Stimulation media was collected from HEK 293 cells or cells stably expressing chicken netrin-1 in starvation media for 3 days. Cells were lysed in mild lysis buffer (MLB) containing protease inhibitor cocktail for 20 mins on ice and lysates centrifuged for 15 mins at 4ºC. To check protein expression, small amounts of cell lysates

15 were aliquoted, mixed with loading buffer, and boiled at 95-100ºC for 5 minutes. For immunoprecipitation, 0.5 μl (anti-flag and anti-DSCAM) or IgG/IgA beads only was added to lysate and incubated 4 hours to overnight at 4ºC. IgG or IgA beads were added for 3 hours, washed 3X in cold MLB, boiled, and separated by SDS-PAGE. After transferring, membrane was blocked with 5% milk and incubated in primary antibody overnight at 4ºC. Membranes were washed 3X with PBST (1X PBS + 0.1% Tween-20) and placed in secondary antibody for 1 hour, washed 3X with PBST, and exposed on autoradiography film (Denville) using ECL (Thermo).

3.4 Dissociated Primary Neuron Culture

The E15 mouse cortex and cerebellum, and the P2-4 mouse cerebellum were dissected in cold HBSS and tissues were dissociated at 37ºC using 0.25% Trypsin for 15 mins and DNaseI for 5 mins. Cells were triturated with ice-cold DMEM culture medium containing 10% Fetal Bovine Serum (FBS) , centrifuged at 1000 RPM, and washed 3X in culture medium before plated at 4x106cells on PLL-coated 35 mm tissue culture dishes for biochemical analysis or 1x104 cells on PLL-coated glass coverslips for the growth cone collapse or immunostaining assays.

3.5 Growth Cone Collapse

The P3-4 mouse cerebellum was dissected and cut into 200 μm sections using a tissue chopper (Vibratome). Only the EGL (external granular layer) was finely dissected using a sharpened tungsten needle. After dissociation, 1x104 cells were nucleofected

16

(Amaxa) with Venus-YFP (1 μg) only or Venus plus 4 μg DSCAM and/or UNC5C constructs using program G-013. Cells were plated in DMEM + B27 + 1X

Penicillin/Streptomycin. After 4 hours, media was replaced to wash away toxic nucleofection solution. After 18h, neurons were starved in DMEM + 0.1% BSA + B27 for 5 hours, stimulated with HEK control or netrin-1 conditioned media+B27 for 30 mins, and fixed in 4% PFA for 15 mins. Images of only YFP-labeled cells were taken using an epifluorescent microscope. Growth cone areas were visualized with Phalloidin (F-actin) and measured using ImageJ (NIH). At least 150 neurons were measured per group and experiments were done in triplicate. Pictures represent merged YFP, Phalloidin, and

DAPI staining.

3.6 Ex vivo Cerebellar Slice Electroporation and Culture

P5-P7 mouse cerebella were dissected and cut into 300 μm sections using a tissue chopper (Vibrotome). Slices were placed in a petri dish electroporator (BTX). Plasmid solution consisted of 1 μg Venus-YFP only or Venus plus 5 μl plasmids, 10X Fast Green

(for visualization), and HBSS. Plasmids were placed next to edge of the cerebellum and electroporated using the following program: 100 V, 50 ms square pulses, 6 pulses with

1.0 s intervals. Slices were immediately transferred to cold HBSS for 10 mins and onto a membrane insert (Millipore) in a 35mm dish containing 1 ml cold DMEM + 10% FBS +

1X Penicillin/Streptomycin. Slices were fixed in warm 4% PFA for 6 hours after culturing 1-3 days. For cultures longer than 1 day, the culture medium was replaced with

1 ml warm fresh medium and changed in alternating days thereafter. After slices were

17 stained and mounted onto slides, images were taken using a fluorescent confocal microscope (Zeiss). For quantification, the distance between the edge of the cerebellum and PCL was divided in half. The outer half is quadrant 1 and the inner half is quadrant 2.

Any cell body in the PCL or IGL is quantified as quadrant 3.

3.7 Cryosection

E15 mouse were collected and placed in cold 4% PFA overnight. P4-P6 mouse pups were perfused with 4% PFA intracardially. All brain tissues were placed in cold 4% PFA for further fixation. Tissues were washed in PBS, transferred to cold 30% glucose overnight, and embedded in OCT. 10 μm coronal slices were cryosectioned and fixed with cold acetone on superfrost plus slides. Slides were post-fixed with 4% PFA for

30 mins, washed in PBS, and subjected to immunostaining.

3.8 Immunostaining

Dissociated Neurons

Neurons on coverslips were permeabilized with 0.1% TritonX-100 for 15 mins and blocked in 10% Normal Goat Serum for 1 hr. Cells were placed in the primary antibody solution for 2 hrs at 37ºC, washed 3X in PBS, and incubated with the secondary antibody for 1 hr at 37ºC. After washing 3X, coverslips were counterstained with DAPI, washed again, and mounted onto slides using Fluorogel.

Slice

18

For studying the expression pattern of DSCAM and UNC5C in the developing nervous system, frozen sections of brain tissues were permeabilized in PBST (1XPBS with 0.3% TritonX-100) for 2 hrs, blocked in 10% Normal Goat Serum for 1 hr and incubated with the primary antibody overnight. After being washed 3X in permeabilization buffer, slices were incubated with the secondary antibody for 2 hrs at

37ºC. For thick culture slices, the permeabilization step was conducted overnight and blocked in 5% Bovine Serum Albumin for 2 hrs. Slices were placed in the primary antibody for 3 days, washed in permeabilization buffer extensively, and incubated with the secondary antibody overnight. Slices were washed again and mounted on slides using

Fluorogel.

19

Chapter 4

Results

4.1 Biochemical Analysis of DSCAM and UNC5C

Interaction

4.1.1 Interaction of DSCAM and UNC5C in transfected

HEK 293 cells

Figure 4-1: DSCAM and UNC5C interaction in transfected HEK 293 cells.

DSCAM interacted with UNC5C (Lane 2) compared to IgA negative control (Lane 1).

To test whether DSCAM interacts with UNC5C in vitro, DSCAM-Flag and

UNC5C-HA were co-transfected in HEK 293 cells. DSCAM was immunoprecipitated with anti-Flag and membrane was blotted for Flag and UNC5C. DSCAM pulled down

20

UNC5C (Lane 2) compared to IgA beads only (Lane 1). Because DSCAM and UNC5C interact in HEK 293 cells, we wanted to know if they interact endogenously in primary neurons.

4.1.2 Interaction of endogenous DSCAM and UNC5C in E15 cortical neurons

Figure 4-2: DSCAM and UNC5C interacted endogenously in E15 mouse cortical neurons. UNC5C interacted with DSCAM (Lane 2) and induction of the interaction by netrin-1 stimulation (Lane 3) compared to HEK stimulation (Lane 2).

Cortical neurons were used because cortex dissection, dissociation, and culturing is fairly easy yielding an abundant amount of neurons. Also, cortical neurons express

DSCAM, UNC5C, and DCC. After dissociating and culturing neurons, primary neurons were serum-starved for 4 hours and stimulated with netrin-1 or control HEK conditioned media for 20 mins. DSCAM was immunoprecipitated with anti-DSCAM and the membrane was blotted for UNC5C and DSCAM. UNC5C interacted with DSCAM (Lane

21

2) and netrin-1 stimulation (Lane 3) increased DSCAM/UNC5C interaction, compared to

HEK stimulation (Lane 2). Next, we wanted to see the temporal pattern of netrin-1 induction on DSCAM/UNC5C interaction. Does netrin-1 induction increase over time and for how long?

4.1.3 Time course of the netrin-1-induced endogenous

DSCAM and UNC5C interaction in E15 cortical cells

Figure 4-3: The interaction of endogenous DSCAM and UNC5C increased after netrin-1 stimulation in E15 mouse cortical cells. In E15 cortical cells, netrin-1 induced endogenous DSCAM/UNC5C interaction up to 15 mins (Lanes 2-5).

Again, E15 cortical neurons were cultured, stimulated with netrin-1 conditioned media, and lysed as previously described. DSCAM was immunoprecipitated with anti-

DSCAM and membrane was blotted for both DSCAM and UNC5C. Netrin-1 enhanced the interaction of DSCAM and UNC5C up to 5 mins and stabilized interaction up to 15 mins. At 20 mins, the DSCAM/UNC5C interaction decreased.

22

Both DSCAM and UNC5C are strongly expressed in the postnatal cerebellum.

From P0-P6, granule cells account for majority of the cerebellum. Granule cells also express UNC5C, mediating netrin-1 repulsion (Przyborski et al., 1998). To study whether

DSCAM interacted with UNC5C in the postnatal mouse cerebellum, we dissociated P2 mouse cerebellar neurons and stimulated them with or without netrin-1.

4.1.4 Time-dependent induction of endogenous DSCAM and

UNC5C interaction in P2 cerebellar neurons by netrin-1

Figure 4-4: DSCAM and UNC5C interaction increased over time with netrin-1 stimulation in P2 cerebellar neurons. DSCAM and UNC5C interacted (Lane 2) compared to IgG beads only (Lane 1). Netrin-1 increased DSCAM/UNC5C interaction persistently over 30 mins (Lanes 2-5).

23

We carried out the same time course method as described previously, but in dissociated P2 cerebellar neurons and stimulated them with netrin-1 up to 30 mins.

DSCAM was immunoprecipitated with anti-DSCAM and blotted for DSCAM and

UNC5C. Interestingly, netrin-1 dramatically induced DSCAM/UNC5C interaction and persistently increased this interaction over 30 mins. Next, DSCAM and UNC5C truncation mutants were transfected into HEK 293 cells to reveal which domains are involved for the interaction.

4.1.5 The Domain Interaction of DSCAM and UNC5C in

HEK 293 cells

Figure 4-5: The DSCAM extracellular domain interacted with UNC5C. The full- length DSCAM (Lane 2) and the DSCAM extracellular domain (Lane 3) but not the intracellular domain (Lane 4) interacted with UNC5C.

24

Figure 4-6: The UNC5C Ig2 domain interacted with DSCAM. The UNC5C Ig2 domain was sufficient to interact with DSCAM (Lane 3) compared to Ig1 (Lane 2) and

Tsps (Lane 5) domains. UNC5C could not interact with DSCAM when both Ig1 and Ig2 domains were truncated (Lane 4).

First, biochemical analysis of DSCAM truncation mutants co-transfected with

UNC5C in HEK 293 cells revealed the DSCAM extracellular domain was necessary for the UNC5C interaction. (Lane 3). Then, UNC5C domain truncations transfected with

DSCAM showed the UNC5C Ig2 domain interacted with DSCAM (Lane 3 and 4).

Therefore, DSCAM and UNC5C interacted through extracellular domains.

4.1.6 RNAi knockdown of UNC5C in transfected HEK 293 cells.

25

Figure 4-7: RNAi knockdown of UNC5C in transfected HEK 293 cells. UNC5C shRNA 5b efficiently reduces UNC5C expression compared to WT. 5b used as UNC5C shRNA and 6b used as UNC5C control shRNA.

For functional assays, we developed UNC5C shRNAs and co-transfected them with UNC5C in HEK 293 cells. After 1 day, cells were lysed and run on gel to check

UNC5C expression. 5b efficiently reduced UNC5C and used in functional assays as

UNC5C shRNA and 6b was used in subsequent assays as UNC5C control shRNA.

Before proceeding to functional assays, we wanted to know the expression pattern of DSCAM and UNC5C in embryonic and postnatal neurons and postnatal brain slices.

Primary neurons from the E15 cortex, the E15 cerebellum and the postnatal cerebellum were immunostained for DSCAM and UNC5C.

4.2 The DSCAM/UNC5C expression pattern in embryonic and postnatal mouse dissociated neurons and brain slices.

26

4.2.1 Co-expression of DSCAM and UNC5C in dissociated

E15 mouse cortical and cerebellar neurons, and P2 cerebellar neurons.

Figure 4-8: DSCAM and UNC5C are coexpressed in dissociated E15 mouse cortical and cerebellar, and P2 cerebellar neurons. DSCAM (A, E, I) and UNC5C (B, F, J) expression in dissociated E15 cortical (A-D), cerebellar (E-H), and P2 cerebellar (I-L) neurons. DAPI staining (C, G, K) labels cell body and merged images (D, H, L) showed the DSCAM/UNC5C colocalization.

Brain tissues from the P2 cerebellum and the E15 cortex and cerebellum were dissociated and cultured. Primary neurons were immunostained for DSCAM (green) and

UNC5C (red). Both DSCAM and UNC5 were expressed and colocalized (yellow) in the cortical and cerebellar neurons. Western blots also confirmed their expression in

27 dissociated neurons. Cerebellar cryosections were also immunostained with DSCAM and

UNC5C to reveal their expression patterns in different layers of P4 cerebellar and cortical slices.

4.2.2 The expression pattern of DSCAM and UNC5C in the

P4 cerebellar and cortical slices

Figure 4-9: The co-expression of DSCAM and UNC5C in P4 cortical and cerebellar slices. Coronal slices of P4 mouse cerebellum (A-C) and cortex (D-F) showing DSCAM

(A, D), UNC5C (B, E), and merged (C, F) images.

28

P4 mouse pups were perfused intracardially with 4%PFA and brains were removed and post-fixed, and prepared for cryosectioning. 10 μm sections were collected and immunostained for DSCAM and UNC5C. In the cerebellum, DSCAM is strongly expressed in the Purkinje cell layer (PCL) and to a lesser extent in the external (EGL) and the inner (IGL) granule layers. UNC5C is expressed throughout the cerebellum and also stronger in the PCL. Previous studies have shown that UNC5C mRNA is strongly expressed in the EGL and the IGL (Alcantara et al., 2000). DSCAM is expressed in neurons within the cortical layers and also at the cortical plate surface. UNC5C is also expressed in the outer most cortical layer.

UNC5C mediates netrin-1 repulsion in EGL cells. Because DSCAM interacts with UNC5C in cerebellar granule neurons, and DSCAM is also co-expressed with

UNC5C in EGL cells, does DSCAM also play a functional role in netrin-1 repulsion?

To test the functional role of DSCAM in netrin-1 mediated repulsion, we adopted an in vitro growth cone collapse assay. UNC5A and UNC5B have been shown to mediate growth cone collapse in netrin-1 signaling (Bartoe et al., 2006; Hata et al., 2009).

UNC5C is involved in netrin-1 repulsion in EGL cells in co-culture studies (Alcantara et al., 2000). UNC5C has not been shown to mediate netrin-1 induced growth cone collapse.

In the growth cone collapse assay, dissociated EGL neurons were subject to netrin-1 or HEK conditioned media bath stimulation. Therefore, netrin-1 was distributed throughout the media (no gradient) and will cause the axon growth cone collapse. Will

EGL neurons growth cones collapse upon netrin simulation?

29

4.3 Netrin-1 induced EGL cell growth cone collapse

4.3.1 The role of DSCAM in EGL cell growth cone collapse

Figure 4-10: DSCAM was required for netrin-1 induced EGL cell growth cone collapse. DSCAM knockdown in EGL cells abolished netrin-1 induced growth cone collapse (I-J) compared to untransfected (A-B), Venus-YFP (C-D), empty shRNA vector

(E-F), or control DSCAM shRNA (G-H). Removing the DSCAM intracellular domain abolished collapse (K-L), while the DSCAM extracellular domain deletion caused partial collapse (M-N).

30

Figure 4-11: DSCAM mediated netrin-1 repulsion in EGL cells. Bar graph representing the figure 4-10. Two-tailed Student’s t-test was performed. * p value <0.001 compared to HEK. # p value <0.05 compared to HEK. Error bars represent S.D.

We first tested whether EGL neuron growth cone collapse in presence of netrin-1.

P4 cerebellar cells were dissociated and cultured, starved for 4 hours, stimulated with netrin-1 or control media for 30 mins, and fixed. Netrin-1, compared to HEK, stimulated

EGL cell growth cone collapse (repulsion) in untransfected (A-B) neurons. First, we wanted to know the role of DSCAM in EGL cell growth cone collapse. Interestingly,

DSCAM knockdown abolished netrin-1 induced growth cone collapse (J) compared to

HEK stimulation (I), DSCAM shRNA control groups (E-H), and Venus-YFP only (C-D).

Using truncation mutants, the DSCAM intracellular domain is necessary for downstream netrin-1 repulsion signaling (K-L). However, the DSCAM extracellular domain also plays a role in growth cone collapse (M-N). DSCAM may need to interact with UNC5C to mediate netrin-1 repulsion.

31

Canonically, UNC5C mediates netrin-1 repulsion in neuronal migration and EGL cell axons are repelled in the presence of netrin-1 aggregates (Przyborski et al., 1998;

Alcantara et al., 2000). To test whether DSCAM or UNC5C plays a dominant role in netrin-1 induced repulsion, the role of UNC5C was dissected using the growth cone collapse assay.

4.3.2 The role of UNC5C in the EGL growth cone collapse

Figure 4-12: UNC5C mediated netrin-1 induced EGL cell growth cone collapse.

UNC5C knockdown in EGL cells abolished netrin-1 induced growth cone collapse,

32 compared to HEK (E-F), opposed to Venus-YFP (A-B) and control UNC5C shRNA (C-

D). UNC5C overexpression resulted in collapse (G-H). The UNC5C intracellular domain

(I-J) but not the extracellular domain (K-L) abolished netrin-1 induced collapse.

Figure 4-13: UNC5C mediated netrin-1 repulsion in EGL cells. Bar graph representing the figure 4-12. Two-tailed Student’s t-test was performed. * p value <0.001 compared to HEK. Error bars represent S.D.

Using the same stage cerebellar cells and stimulation methods, we investigated whether UNC5C also mediated growth cone collapse in EGL neurons. UNC5C knockdown blocked the growth cone collapse in netrin-1 bath incubation (F) compared to

HEK stimulation (E) and control groups (A-D). Further analysis revealed the UNC5C intracellular domain was necessary for the netrin-induced growth cone collapse (I-J) compared to the partial collapse when the UNC5C extracellular domain is truncated (K-

L). UNC5C and DSCAM extracellular domains played partial roles in growth cone

33 collapse, suggesting the interaction of these receptors plays a role in mediating netrin-1 induced collapse in EGL neurons.

Both DSCAM and UNC5C mediate netrin-1 repulsion in EGL cells. Next,

DSCAM and UNC5C were knockdown to test the function of netrin-1 induced growth cone collapse.

4.3.3 The combined role of DSCAM and UNC5C in netrin- induced growth cone collapse

Figure 4-14: Simultaneous knockdown of DSCAM and UNC5C abolished netrin-1 induced EGL cell growth cone collapse. DSCAM (C-D) or UNC5C (E-F) knockdown blocked netrin-1 induced collapse compared to Venus-YFP (A-B). Simultaneous

DSCAM and UNC5C knockdown abolished netrin-1 induced collapse (I-J) compared to control shRNAs (G-H).

34

Figure 4-15: Simultaneous knockdown of DSCAM and UNC5C blocked netrin-1 induced growth cone collapse in EGL cells. Bar graph representing the figure 4-14.

Two-tailed Student’s t-test was performed. * p value <0.001 compared to HEK. Error bars represent S.D.

To study the coordinating role of DSCAM and UNC5C in netrin repulsion, both

DSCAM and UNC5C were knockdown to reveal netrin-1 effect on growth cone collapse.

As expected, simultaneous knockdown of DSCAM and UNC5C blocked netrin-1 collapse. This result was expected since DSCAM or UNC5C alone also abolished collapse, suggesting DSCAM and UNC5C interaction coordinates netrin-1 induced growth cone collapse.

In an in vitro assay, other guidance mechanisms are removed from the system as neurons can act in a stimulation environment or autonomously. In explants or tissues slices, neurons may gain function through other signaling mechanisms from nearby

35 neurons or cells. Because DSCAM and UNC5C mediated netrin-1 repulsion in an in vitro system, we wanted to test their function in an in vivo system. Postnatal cerebellum slices were used in culture to study neuronal migration patterns. UNC5C deficient mice reveal ectopic granule cell migration patterns, suggesting netrin/UNC5C repulsion is lost

(Przyborkski et al., 1998). In co-culture studies, EGL cells are seen migrating out and away from explants co-cultured next to netrin-1 aggregates, also suggesting UNC5 mediates netrin-1 repulsion in neuronal migration (Alcantara et al., 2000). These studies suggest that UNC5C mediating netrin-1 repulsion may be involved in EGL cell migration.

We adopted the ex vivo electroporation and slice culture using P5-7 cerebellum slices. The cerebellum was dissected, cut into slices, electroporated with specific constructs, and cultured on membrane inserts. DSCAM and UNC5C mediating netrin-1 repulsion can be studied in close to an in vivo system as possible.

36

4.4 The ex vivo cerebellar slice electroporation and culture assay

Figure 4-16: DSCAM knockdown increased EGL cell migration rate after 2 DIV.

DSCAM knockdown increased neuronal migration rate (C-D) compared to WT (A-B) after 2 DIV.

37

Figure 4-17: UNC5C knockdown slowed migration rate while both DSCAM and

UNC5C knockdown increased neuronal migration rate. UNC5C knockdown decreased migration rate (E-F) while simultaneous DSCAM and UNC5C knockdown resulted in increased migration (G-H) compared to Venus (Figure 4-16 A-B) after 2 DIV.

38

Figure 4-18: Knockdown of DSCAM and UNC5C did not change migration rate significantly in EGL neurons after 1 day in culture. Figure 33-34 representative data

(A, C, E, G). Two-tailed Student’s t-test was performed. * p value <0.05 compared to

Venus. No significant difference comparing similar quadrants from different groups.

39

Figure 4-19: Knockdown of DSCAM and UNC5C changed migration rate in EGL neurons after 2 days in culture. Figure 33-34 representative data (B, D, F, H). Two- tailed Student’s t-test was performed. * p value <0.05 compared to Venus. Similar quadrants were compared to different groups.

After electroporation, slices were cultured for 1-2 days. After fixation, green labeled cells were visualized in slices counterstained with anti-calbindin, the Purkinje cell layer (PCL) marker. After DSCAM knockdown or simultaneous DSCAM and UNC5C knockdown, more EGL neurons were in quadrant 2 and 3 compared to Venus after 2 days. The difference was not evident after only 1 day slice culture. UNC5C deficient neurons still migrated away from netrin-1 but migrated ectopically from IGL into WM, suggesting netrin-1 repulsion was lost (Alcantara et al., 2000). DSCAM deficient neurons

40 migrate faster towards the IGL, suggesting netrin-1 repulsion is lost and these cells may too migrate aberrantly.

More strikingly, UNC5C knockdown neurons accumulated in the EGL layer, compared to Venus, after 2 days. Loss of repulsion caused these cells to migrate slowly, contradicting the role of DSCAM in mediating netrin-1 repulsion in vitro. DSCAM and

UNC5C mediating netrin-1 repulsion in EGL cell migration and axon guidance may involve other guidance mechanisms. DCC mRNA is strongly expressed in EGL cells

(Alcantara et al., 2000). Canonically, DCC aids in UNC5C mediated long-range netrin-1 repulsion. Also, Slit/Robos signaling is involved in EGL neuronal repulsion. In our lab, we have also shown that DSCAM interacts with Robo1 (unpublished data).

In conclusion, in vitro and slice culture phenotypes revealed a new role for

DSCAM in mediating-netrin-1 induced repulsion in EGL neurons.

41

Chapter 5

Discussion

Previous studies indicate that DSCAM functions as a netrin-1 axon guidance receptor in commissural axons (Andrews et al., 2008; Liu et al., 2009; Ly et al.,

2008). However, other evidence also suggests DSCAM may function in netrin-1 repulsion. DSCAM mRNA is expressed in cerebellar EGL and IGL neurons. In these neurons, expression of UNC5C and netrin-1 is high (Alcantara et al., 2000). UNC5C deficient granule cells migrate ectopically from netrin-1 source into the midbrain regions and the cerebellar white matter (WM) (Przyborski et al., 1998; Alcantara et al., 2000). Ectopic migration into the midbrain regions can result from functional loss of netrin-1 repulsion. EGL cells migrate into the IGL but cannot repel away from the netrin-1 source located in the fourth ventricle zone, suggesting netrin-

1/UNC5C long-range repulsion is lost. DSCAM is expressed in the EGL and the

IGL, suggesting a functional role in netrin-1 repulsion. Genetic and medical evidence also provide clues for DSCAM function in the cerebellum. dscam lies within the extra copy of chromosome 21 region associated Down syndrome (Yamakawa et al.,

1998). Down syndrome patients have a smaller cerebellum, possibly leading to cognitive defects associated with improper wiring of the cerebellum circuitry. These phenotypes could be a possible result of overexpression of DSCAM. Therefore, we

42 hypothesized that 1) DSCAM may interact with UNC5C, 2) DSCAM functions in netrin-1 repulsion in the cerebellum.

First, we tested whether DSCAM interacted with UNC5C. DSCAM and

UNC5C interacted in transfected HEK 293 cells. More importantly, DSCAM and

UNC5C interacted endogenously in E15 cortical neurons and netrin-1 stimulation enhanced the interaction. In HEK 293 cells, UNC5C and DSCAM interact without any netrin-1 stimulation. However, in E15 cortical cells, UNC5C and DSCAM interaction is subtle even before netrin-1 stimulation. In a netrin-1 stimulation time course, DSCAM and UNC5C interaction was weak at 0 mins, increased at 5 mins and induction was maintained up to 15 mins. In HEK 293 cells, we overexpressed these proteins transiently and DSCAM and UNC5C were in close proximity to each other on the membrane surface, allowing for basal level interaction. Transfecting

DSCAM and UNC5C in HEK 293 cells and seeing interaction at basal level suggests that these two proteins may also bind directly because some neuronal proteins may not be expressed in HEK 293 cells. Direct binding with purified proteins can confirm this hypothesis.

Previous studies have shown that UNC5C, DSCAM, and DCC mRNAs are all expressed in embryonic cortex and netrin-1 increases cortical axon outgrowth and attraction. The attractive effect of netrin-1 on cortical axon projection depends on high expression of DCC and DSCAM and low levels of UNC5C. Next, we tested the interaction of DSCAM and UNC5C in cultured P2 cerebellar neurons because

UNC5C mediates netrin-1 repulsion in cerebellar granule cells and a majority of the cerebellum contains granule cells from P0 to P6 (Przyborski et al., 1998; Alcantara et

43 al., 2000). Strikingly, endogenous DSCAM interacted with UNC5C and netrin-1 dramatically increased this interaction, suggesting that DSCAM and UNC5C interaction plays a crucial role, mediated by netrin-1, in the developing cerebellum.

Quite possibly, netrin-1 binding to either DSCAM or UNC5C can cause a conformational change at the extracellular domains of these receptors, allowing for interaction if in close proximity. In cortical cells, DSCAM mediates netrin-1 attraction, explaining the weak netrin-1 induction on the interaction. In cerebellar cells, netrin-1 caused the dramatic association between the two receptors, mediating netrin-1 repulsion.

To further dissect the interaction, both DSCAM and UNC5C truncation mutants were transfected in HEK 293 cells, revealing the DSCAM extracellular domain and the UNC5C Ig2 domain are sufficient for this interaction. The truncation mutants provided insight on how exactly DSCAM and UNC5C may associate before and after netrin-1 stimulation. In neurons, before netrin stimulation, DSCAM and

UNC5C interact at the basal level. Upon netrin-1 stimulation, conformational state of

DSCAM and UNC5C extracellular domains change, allowing both proteins to interact. Intracellular signaling mechanism will be crucial in determining netrin-1 signaling mechanism downstream of both receptors. Quite possibly, netrin-1 can bind to one receptor, allowing the growth cone to sense a netrin-1 gradient, re-localizing both receptors into close proximity, and enhancing netrin-1 signaling. Many possibilities can exist on whether netrin-1 binds to one receptor or the other, or maybe both. More importantly, how do downstream proteins of both receptors coordinate netrin-1 repulsion? Biochemical analysis revealed a novel interaction

44 between netrin-1 receptors UNC5C and DSCAM through their extracellular domains.

The data suggests that DSCAM could be involved in netrin-1 repulsion.

Immunostaining of dissociated cortical and cerebellar neurons reveals co- expression and co-localization of DSCAM and UNC5C throughout the primary cortical and cerebellar neurons. Both proteins are present at the tip and in the axon.

Although the role of UNC5C in dendrites is not known, DSCAM is involved in dendritic arborization and synapse formation. In P4 cerebellar slices, DSCAM is located in the EGL and the IGL layers inhabited by granule cells, compared to the strongest staining in PCL. UNC5C is expressed throughout the cerebellum slice as well. Our slice expression data coincide with mRNA expression patterns (Alcantara et al., 2000). Co-localization of DSCAM and UNC5C in granule cells suggests

DSCAM may collaborate with UNC5C involved in netrin-1 repulsion.

Hata et al. and Bartoe et al. provided evidence of UNC5 family members mediating growth cone collapse in different neurons, such as overexpression of

UNC5A in hippocampal neurons and UNC5B in cortical neurons. (Hata et al., 2009,

Bartoe et al., 2006). EGL explant axons projected away from netrin-1 aggregates in co-culture, suggesting netrin-1 repulsion (Alcantara et al., 2000). Therefore, we adopted the in vitro growth cone collapse assay to test whether netrin-1 stimulation on EGL neurons can cause growth cone collapse. Only the EGL layer from postnatal cerebellar slices was dissected, dissociated, and cultured; hence, the in vitro assay simplifies the surrounding EGL neuronal environment.

We first stimulated cultured EGL neurons with netrin-1 and measured growth cone area. Interestingly, netrin-1 caused EGL neuron growth cones collapse, even

45 without overexpression of UNC5 in these neurons as previous studies described.

Because UNC5C is known to mediate netrin-1 repulsion in EGL cells, we first wanted to see if DSCAM plays any role in mediating collapse. Surprisingly, DSCAM knockdown blocked growth cone collapse in the presence of netrin-1. This suggested that DSCAM plays a role in netrin-1 induced repulsion in EGL neurons. Further analysis revealed that the DSCAM intracellular domain was necessary to transduce netrin-1 signal, suggesting that netrin-1 repulsion signal is mediated through

DSCAM. DSCAM intracellular domain truncation (DSCAM∆C) is a dominant-negative form of the protein. Initially, when DSCAM∆C was transfected into proteins, the growth cones were larger than untransfected EGL neurons (Data not shown), suggesting

DSCAM may play a cell-autonomous role in maintaining growth cone size. The larger growth cones may be more sensitive to netrin-1. Also, DSCAM∆C can disrupt proper downstream signaling when bound to netrin-1 and interacted with UNC5C, blocking repulsion. From the growth cone collapse assay, intracellular domain of DSCAM was necessary to mediate netrin-1 repulsion. DSCAM∆C could play other aberrant functional roles that ultimately disrupt growth cone maintenance. DSCAM is a cell adhesion molecule and may interact with other integrins or cell adhesion molecule family members, playing a crucial role in mediating netrin-1 growth cone collapse.

Next we dissected the role of UNC5C in growth cone collapse. We found that

UNC5C knockdown abolished netrin-1 induced growth cone collapse, suggesting that

UNC5C also plays a role in netrin-1 repulsion. Like DSCAM, the UNC5C intracellular domain is important for collapse and the extracellular domain partially blocks collapse, proposing that the UNC5C intracellular domain can transduce the netrin-1 signal but

46 repulsion is enhanced upon UNC5C interaction with other proteins at the receptor level or coordination through downstream signaling cascades.

To test the function of both UNC5C and DSCAM, co-knockdown of both proteins in EGL neurons resulted in loss of netrin-1 induced growth cone collapse. This result was expected since DSCAM or UNC5C alone can mediate growth cone collapse. However, both domain truncation phenotypes suggest that UNC5C and DSCAM coordinate with each other on the growth cone surface, or other downstream signaling cascades to mediate netrin-1 repulsion. To convert DCC attraction to repulsion, UNC5B binds to the intracellular domain of DCC. Also, netrin-1 only needs to bind to DCC but the repulsion signal is transduced by the intracellular domain of UNC5B (Hong et al., 1999). DSCAM and UNC5C interacted through their extracellular domains. According to growth cone collapse, netrin-1 can interact with either DSCAM or UNC5C to induce collapse because extracellular domain deletion of either protein resulted in partial collapse. These results suggest the coordinating role of DSCAM and UNC5C in netrin-1 mediated axon repulsion.

How can both proteins mediate netrin-1 repulsion individually? Netrin-1 can interact with either DSCAM or UNC5C and their downstream signals may cross-talk, ultimately mediating netrin-1 repulsion. Therefore, downstream signaling probably plays the most crucial role in coordinating growth cone collapse by netrin/DSCAM and netrin/UNC5C. Previous studies suggested that FAK and Src kinases are involved in netrin-1/UNC5C signaling. In our lab, we have shown that Pak1 and Fyn interact with the

DSCAM and UNC5C complex (Data not shown), suggesting these two receptors coordinate downstream signaling events to mediate netrin-1 repulsion. To further dissect

47 the downstream signal cascades of netrin/DSCAM/UNC5C, EGL cells will either be nucleofected with FRNK (dominant negative form of FAK), Pak1 siRNA, or stimulated with PP2 (Src family kinases inhibitor), and PP3 (a control of PP2) in the presence of netrin-1 to assess the role of these downstream signaling molecules in growth cone collapse. Also, biochemical analysis of downstream proteins activated by either UNC5C or DSCAM can dissect the potential mechanism leading to netrin-1 repulsion.

To further assess the role of DSCAM in netrin-1 repulsion, we wanted to use an in vivo system where neurons function in their natural environment. In the developing cerebellum, EGL neurons migrate away from netrin-1 expressed in the EGL (Alcantara et al., 2000). Neurons migrate out and away from EGL explants cultured next to netrin-1 aggregates, suggesting netrin-1 repulsion in neuronal migration (Alcantara et al., 2000).

We adopted the ex vivo slice electroporation and culture system to study the role of

DSCAM and UNC5C in EGL neuronal migration patterns. Briefly, the cerebellum was dissected and cut into slices. Each slice was electroporated with specific constructs and cultured on membrane inserts in culture medium for 1 or 2 days. The slice acts as the in vivo environment, allowing the function of DSCAM and UNC5C in EGL neuronal migration to be evaluated.

We first electroporated Venus-YFP only into EGL neurons. After immunostaining the slices with the PCL marker, migration patterns of EGL cells were observed. In the

EGL, granule cells project two horizontal processes while migrating towards the ML. In the ML, granule cells will project a third axon vertically, attaching to Bergmann glial fibers and pulling the soma down into the IGL.

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After 1 day in vitro (DIV) slice culture, YFP labeled EGL neurons migrated to the lower EGL because many neurons had only horizontal axonal projections. After 2 DIV, more neurons (compare to 1 DIV) were seen in the ML and some even in the IGL (pass the PCL marker) because these neurons project an axon vertically downward towards the

IGL layer. When knockdown of DSCAM only or both DSCAM and UNC5C in EGL neurons using RNA interference, after 1 DIV, some cells already projected axons into the

IGL. More prominently, more EGL neurons were located in the ML and the IGL compared to YFP only group, suggesting that DSCAM may play a dominant role, compared to UNC5C, in migrating neurons repelled by netrin-1. Both DSCAM and

UNC5C knockdown neurons also migrated faster towards the IGL. DSCAM signaling may ultimately guide the neuron away from the netrin-1 source in the outer EGL.

Mechanistically, netrin-1 can bind to either DSCAM or UNC5C, causing enhancement of

UNC5C/DSCAM interaction. Then, UNC5C and DSCAM downstream signaling coordinates specific signaling events, mediating repulsion. Interestingly, phenotypes of

DSCAM deficient granule cell migrations are similar to those in UNC5C knockout mice.

These data indicate that both DSCAM and UNC5C are involved in netrin repulsion.

Another possibility is other guidance mechanisms may play a role in coordinating netrin/DSCAM signaling. DCC is strongly expressed in the outer EGL and could exacerbate repulsion in granule cells by binding to UNC5C only. This raises the question of exactly what role does DCC play in EGL neuronal migration? Does DCC also mediate repulsion in granule cells by binding to UNC5C or does DCC mediate netrin-1 attraction, balancing or opposing netrin-1 repulsion in EGL cell migration? Also, Slit is strongly expressed in the EGL layer as well, mediating repulsion in granule cell migration. In our

49 lab, we have identified a novel interaction between DSCAM and ROBO (unpublished data), suggesting a complicated role of DSCAM mediating different mechanisms in cell migration. Therefore, many layers of mechanisms play distinct roles in guiding granule cell migration. The key is to understand how all these possible mechanisms work together or antagonize each other, mediating granule cell migration from the EGL to the IGL.

In conclusion, 1) DSCAM interacts with the netrin-1 repulsive receptor UNC5C and netrin-1 stimulation increases this interaction; 2) DSCAM mediates netrin-1 repulsion in vitro and in ex vivo; 3) DSCAM may coordinate with UNC5C in netrin-1 mediated repulsion. Thus, our data suggests a new role of DSCAM in netrin-1 repulsion and possible mechanistic explanations into abnormal cerebellar neuronal migrations seen similarly in patients with Down syndrome.

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Chapter 6

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