SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL

SCIENCES AND TECHNOLOGY

THIRUVANANTHAPURAM- 695 011, INDIA

(An institute of National Importance under Govt. of India)

Expression profile of NMDAR and Synaptotagmin in early

stages of neuronal development in PC 12 cell model system

Thesis submitted for the degree of Master of Philosophy in Biomedical technology

MANJULA P M

DECLARATION

I, Manjula P M, hereby declare that I personally carried out the work depicted in the thesis

entitled “Expression profile of NMDAR and Synaptotagmin in early stages of neuronal

development in PC 12 cell model system” under the direct supervision of Dr. Anoopkumar

Thekkuveettill, Scientist F, Molecular Medicine Division, Biomedical Technology Wing, Sree

Chitra Tirunal Institute for Medical Science and Technology, Trivandrum, Kerala, India.

External help sought are acknowledged.

Manjula P M

CERTIFICATE

This is to certify that the dissertation entitled “Expression profile of NMDAR and

Synaptotagmin in early stages of neuronal development in PC 12 cell model system”

submitted by Manjula P M in partial fulfilment for the Degree of Master of Philosophy in

Biomedical Technology to be awarded by this institute. The entire work was done by him under my

supervision and guidance at Molecular Medicine Division, Biomedical Technology Wing, Sree

Chitra Tirunal Institute for Medical Science and Technology (SCTIMST), Thiruvananthapuram 695

012.

Place: Thiruvananthapuram

Date: 25/7/2014 Dr.Anoopkumar

SYNOPSIS

Neurons communicate through a series of functional network between them called

synapses. Synapse formation is one of the most challenging processes to study in vivo. PC12

cell, which can undergo differentiation to neurons in the presence of neurotrophic factors, is one

of the good in vitro models to study this pathway. However it is not clear how early synaptic

connections are established in PC12 cells. One way to study this is to observe the neuronal

growth and do Sholl analysis to measure the dendritic and axonal branching. Besides, the level of

the synaptic specific proteins and their localization also will give a strong insight into the

development of network. In the present study we studied both the Sholl analysis and the

expression pattern of NMDA receptor (post synaptic protein) and synaptotagmin (presynaptic

protein) in developing PC12 cells.

Chapter one discusses the background of the study and the literature related to the study.

In the process of synaptogenesis new synaptic connections are formed, the older one eliminated

or stabilized based on the signal it receives. The synapses can be of different types such as

excitatory, inhibitory, electrical, chemical, en passant or terminal synapses. On the pre synaptic

terminal, synaptotagmin functions as the Ca2+ sensor for the exocytosis of synaptic vesicles. The

protein is composed of N terminal domain, trans membrane region and cytoplasmic domain-C2A

and C2B. On the post synaptic terminal, NMDA receptor is involved in synaptic plasticity, the

process of learning and memory. It has three subtypes –NR1, NR2 and NR3. NR1 includes 8

splice variants. The protein consist of the extracellular amino- terminal domain (ATD), the

extracellular ligand-binding domain (LBD),the trans membrane domain (TMD), and an

intracellular carboxyl-terminal domain (CTD). The differentiation of PC 12 cells by NGF is

mediated through Trk signaling.

Chapter two explains the materials and methods of the study. The PC12 cells were

differentiated for 6 days by the addition of NGF (nerve growth factor). The sprouting was

observed on each day after NGF addition. Neurite length was measured using image J software

and a single neuron from each day of NGF treatment was traced using Fiji Plugin Simple neurite

tracer, which is used for Sholl analysis to know the complexity of neuron on differentiation. The

formation of neuronal network was visualized by live cell imaging. To know the localization and

distribution of NR1 and Syt1, immunocytochemistry was performed for both undifferentiated

PC12 cells and PC12 treated with NGF for different time periods.

Chapter three explains the results of the experiment and discussion. Neuritic sprouting

was observed with 24 hours of NGF treatment. Extensive interconnecting neurites were observed

by the 6th

day. The results show that there is a progressive increase in the neurite length. The

complexity of neuron was found to be increased in Sholl analysis. The neurite length was

showing a linear pattern of growth. There is a gradual increase in number of branching on day 5

and day 6. The sub branching also showed similar pattern. The length of dendrite and axon,

shows a linear pattern of change, showing a gradual increase. The proteins NR1 and Syt1 are

critical in development of stable synaptic connection. Immunocytochemical analysis shows that

synaptotagmin was distributed presumably on the axon. NR1 was observed both on

undifferentiated and differentiated cells, on differentiation it was found to be distributed to the

dendritic spines in day 6.

Chapter four explains the summary and conclusion of the study. The present study

shows that on early neuronal development, as the neural network forms, there is dramatic change

in the pattern of distribution of NMDA receptor and synaptotagmin. NMDA receptor is

expressed both on undifferentiated and differentiated PC12 cells. The study shows that PC12

cells are appropriate model system for studying neuronal development as well as

neurodegenerative diseases.

CHAPTER 1- INTRODUCTION

1.1 Background

In the highly complex nervous system, the neurons are interconnected to form

the functional synapses. The pre and post synaptic membrane should be in right physical

chemistry for the formation of functional synapses. PC (pheochromocytoma) 12 cell line is

commonly used as an early neuronal developmental model, as they undergo neuronal

differentiation in the presence of neuronal growth factors. This is an ideal model system to study

the neurite growth and molecular pathways involved in synapse formation. Body of work on

NMDA receptors in differentiating PC 12 cells, have shown that there is difference in mRNA

expression levels and the protein levels of NMDA receptor subunits. For example NR1 mRNA

expression was unchanged while the protein expression showed a gradual increase as the cells

differentiated into neurons (Casado et al, 1996). NR1- 2a and NR1-4a, two NR1 splice variants,

are highly expressed in PC12 cells. There is a report that different source PC12 cells have

variations in NR1 expression levels (Edward et al, 2007). In addition to NR1, PC12 cells express

other NMDA receptor subtypes such as, NR2C and NR2D (Edward et al, 2007). However, there

are reports that NR1 mRNA expression could be detected in PC12 cells but the protein

expression level is negligible (Sucher et al, 1993). Since NR1 is critical molecule at the post

synaptic terminal and has an essential role in functional synapse formation, the present study,

which focuses on the expression of NMDA receptor subtype NR1 on early neuronal

development by taking PC12 cell as the model system.

At the pre-synaptic terminal, SNARE complex pathway establishes an active zone at

the axonal terminal and involved in the formation of a function synapse. In the SNARE complex,

Synoptotagmin1 (Syt1) is one of the most essential functional molecule, which act as the Ca2+

sensor. Syt1 level was found to increase during early development and persist in adulthood in rat

CNS (Berton et al, 1996). Previous studied from our lab showed that, soluble Syt1 peptide in the

cytoplasm can regulate the translation and maintain a constant level of Syt1 expression in the

synapse, by interacting with 3’UTR of its own mRNA (Sunitha, 2008). The laboratory has also

showed that PC12 cells with Syt1 over expression, has elongated axons (Sreethu, 2012). Since

there is no work on expression levels of Syt1 in PC12 cells, the present work also attempted to

study the levels and localization of the protein during early development of neurites.

1. 2 Review of Literature

In the human body the nervous system is one of the highly complex system,

mainly composed of neurons and glial cells. During embryonic development the nervous system

originates from ectoderm (de Vellis and Carpenter , 1999) in the form a neural tube, which

further differentiates to both peripheral and central nervous system. Neurons are highly polarized

cells having cell body, dendrites and axons as the major structural parts. Cell body consists of

nucleus and other cellular organelles. Axon and dendrites arise from the cell body which is

mainly involved in the cellular communication (Gilbert, 2010).

1.2.1 Neuronal network & synapse formation

The neurons in the nervous system are interconnected to form functional

networks, which is essential for proper inter cellular communication. This network of

communication is developed by neuritic connections at specialized macromolecular junctions

known as synapses. The synapse are often formed by axons and dendrites which undergo

constant modification, also known as synaptic plasticity, based on the experience and learning of

the individual neurons or by the network of neurons (Martin et al, 2000) . The synaptogenesis,

involve a series of interactions between ligand and receptor, F-actin remodeling, intracellular

signaling pathways. Synapses could be excitatory or inhibitory based the neurotransmitters

involved; for example the glutamate act as an excitatory signal at the synapse and GABA act as

inhibitory. (Shen and Cowman, 2009).

Synaptic prepatterning-

The contact between pre and post synaptic terminals are initiated by the dynamic

movement of dendritic filopodia. The axonal growth, which is enhanced by fusion of synaptic

vesicles to the growth cone of the axonal terminal, guides the dendrite filopodia to interact to

form the early synapse (Sabo et al, 2006). The major cytoskeletol protein in the growth cone of

axons is F actin which is formed by the polymerization of G actin (Chia et al, 2014). This

process of polymerization is mainly within the subareas of the growth cone. The cell adhesion

molecules (CAMs), such as neuroligins and neurexin are also involved in the initial selection of

pre and post synaptic partners (Sudhof 2008). Functionally different synapses found to have

variations in cell adhesion molecules. For example neuroligin1 (NLGN1) is localized at

excitatory synapses whereas neuroligin 2 (NLGN2) is found at inhibitory synapses (Chubykin et

al, 2007 and Tabuchi et al, 2007).

Filopodial Motility-

During the early development of neuron, dendritic filopodia will be abundant.

Their numbers decrease on neuronal maturation and stable dendrites develop dendritic spines,

which are morphologically stable structures (Fiala et al, 1998). The dentritic filapodia are highly

motile in nature hence allowing axonal interactions for development of synapse. The guidance

molecules in the axon like BDNF and EphrinB appear to control motility of dendritic filopodia

once both pre and post synaptic contacts are established (Shen and Cowan, 2009).

The interaction between the brain-derived neurotrophic factor (BDNF) and

tropomycin-related kinase B (TrkB) receptor is mediated through PI 3 kinase signaling.

Generally, BDNF/TrkB signaling activate two guanine nucleotide exchange factors (GEF)-Vav2

and Tiam1, which are involved in receptor trafficking and growth of spine (Miyamoto et al,

2006). In mature glutamatergic synapses, plasticity is regulated by restricting BDNF/TrkB

signaling (Huang and Reichard, 2003). However the exact connection between BDNF/TrkB

signaling in F actin remodeling and filopodial motility is unknown ( Fig:1. A). Besides, TrkB,

Eph B also participate in controlling filopodial motility (Shen and Cowan, 2009).

Once the synaptic contact is established, stabilization of axon and dendrite

connection is initiated by the interaction between ephrinB (present on pre synaptic terminal)

EphB (present on post synaptic terminal) ( Kayser et al, 2008). On the post synaptic membrane,

extracellular domain of EphB2, found to recruit NMDA receptors to the post synaptic terminal.

This recruitment of NMDA receptor increase the spine growth and synapse formation (Dalva et

al, 2000). The interaction between ephrinB and EphB2 believed to facilitate F actin remodeling

(Scshubert and Dotti, 2007) ( Fig: 1. B)

Contact stabilization –

Though there are so many early contacts between dendrite and axons, a selected few

only become stabilized. This selection is mediated through the combined involvement of cell-

surface expressed proteins, such as ephrinB/EphB, Cadherins, SynCaM, and neurexin/neuroligin

(Shen and Cowan, 2009). Besides other cell adhering molecules such as integrin, protocadherins,

neural cell-adhesion molecule (NCAM), apCAM), nectin, L1, fasciclin II , DsCAM , syndecans,

sidekicks etc are also found to play a critical role ( Murthy and Camilli, 2003)( Fig: 1).

Synaptic Maturation-

As a part of synaptic maturation, the trans-membrane receptors, synaptic vesicles,

docking protein, ion channels, mitochondria, scaffolding proteins etc must be targeted to the

synaptic terminals. For example, EphrinB/ EphB2 signaling recruits NMDA receptor to the post

synaptic site (Dalva et al, 2000). Wnt-7a, leads to the clustering of synapsin 1, plays an

important role in pre synaptic maturation (Hall et al, 2000). Kalirin-7 (Rac-GEF), a downstream

molecule of N-Cadherin, is involved in the EphrinB/ EphB pathway and plays a role in spine

maturation (Penzes et al, 2003; Xie et al, 2007).

Figure 1: Signaling in synaptogenesis (A) Model for BDNF/TrkB signaling in

synaptogenesis (B) Model for EphrinB/ EphB2 receptor functions and signaling in

synaptogenesis (Shen and Cowan, 2010).

1.2.2 Types of synapses

Synapses can be categorized as terminal synapses and en passant synapses based

on where the synapses are located within the axon. Terminal synapses are found at the end of the

axon. En passant synapses are located along the axon and can also be found far away from axon

terminal. Both types of synapses are found in vertebrate and invertebrate nervous system (Shen

and Cowan, 2009). Synapses are also classified based on the mode of transmission- chemical

synapses and electrical synapses. Chemical synapses communicate via the release of chemical

messenger, called neurotransmitters, which act as a ligand and bind to specific receptors present

at post synaptic membrane. Electrical synapses allow more rapid way of transmission of ions

across the membrane through ion channels at gap junctions, mainly (Hu and Bloomfield, 2003;

Eccles, 1982). Functionally synapses are classified into excitatory and inhibitory. Excitatory

synapses are mainly glutamatergic in which glutamate is the neurotransmitter, which is binds to

NMDA (N Methyl D Aspartate) receptor or AMPA (α-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid) receptor located on postsynaptic terminals (Sobolevsky and Rosconi et

al, 2009). Inhibitory synapse includes are mainly GABAergic in which γ-Aminobutyric acid

(GABA) is the neurotransmitter and its binding to GABA receptors results in hyperpolarization

of post synaptic membrane leading to inhibitory post synaptic potential (IPSP) (Miller &

Aricascu, 2014).

1.2.3 Pre-synaptic terminal

The pre synaptic terminal is known as the active zone, as it can functionally

influence the connected partner neuron. Due to the presence of docked vesicles and scaffolding

proteins it has a dense appearance. Scaffolding proteins is involved in the recruitment of

synaptic vesicles to the active zone and regulate release of neurotransmitter from the vesicles

(Pfenninger et al, 1969). The main synaptic vesicle release is controlled by SNARE complex

machinery, which involves both membrane and vesicular proteins. SNARE complex allows the

vesicles to dock at the active site and release the neurotransmitters as an action potential arrives.

The whole process is highly choreographed by a series of protein-protein interaction (Dieck et al,

1998; Fenster et al, 2000; Wang et al, 1999).

The trafficking cycle of synaptic vesicle in the axonal terminals involves sequential

steps such as transport of neurotransmitters into the synaptic vesicles, agglomeration and

docking of synaptic vesicles at the active zone, vesicular priming, Ca2+ mediated vesicular

fusion and exocytosis. Once the vesicles releases neurotransmitters, they undergo endocytosis

in three different ways: the vesicles either remain at the active zone for refilling (kiss-and-stay)

or are recycled locally and filled later (kiss-and-run), and a slower pathway that involves

clathrin-mediated recycling and filling later (Sudhof, 2004) (Fig: 2).

Figure 2: Model for synaptic vesicle trafficking ( Sudhof, 2004).

1.2.3.a. Synaptotagmin

Synaptic transmission is one of the fasted cellular responses, and is believed to be

mediated through Ca ions. When an action potential reaches the axonal terminal, there is flux of

Ca2+ which triggers synaptic vesicle exocytosis. The protein synaptotagmin, located on the

synaptic vesicle, found to act as the Ca2+ sensor. Though there are more than sixteen Syt

isoforms have been identified, major functionally active Syts in brain are Syt-1, IV, VII and X

All the isoforms have highly conserved structure (Sudhof 2004).

Structural organization of Syt -

Synaptotagmin protein is a membrane protein having single trans membrane domain,

a short N-terminal region and a C-terminal cytoplasmic domain containing highly conserved two

C2-domains (C2A and C2B) (Perin et al, 1990). The C2-domains of are composed of a stable β-

sandwich containing eight β-plated sheets with flexible loops emerging from the top and bottom

(Fernandez et al, 2001). The C2A domains generally bind three Ca2+ ions, whereas C2B

domains bind only two Ca2+ ions. (Ubach et al, 1998, Sudhof 2013) (Fig: 3).

Fig 3: A. Different isoforms of synaptotagmin B. Structural arrangement of C2 domain of

synaptotagmin (Sudhof, 2013).

Function of Syt-

Synaptotagmin I, II, and IX are found to be involved in the release of neurotransmitter

in Ca2+ dependent manner. Among these the Syt1 is the well studied. The two C2-domains of

Syt1 functions cooperatively for Ca2+ binding. The C2-domains bind to phospholipids in a Ca2+

dependent manner and they also interact with SNARE proteins and syntaxin (Sudhof, 2013).

Synaptic vesicle fusion is mediated by fusion machinery at the presynaptic terminal

containing two components: synaptic SNARE proteins and SM proteins. The SNARE proteins

includes SNAP-25, syntaxin 1 and synaptobrevin. The SNAP-25 and syntaxin which is present

on the presynaptic plasma membrane that form complex with synaptic vesicle protein

synaptobrevin. The SNARE proteins that execute and provides the energy for fusion, but

Munc18-1 and other SM-proteins effectively catalyzing fusion. Chaperons such as cysteine

string protein and synucleins are involved in SNARE complex assembly (Sudhof and Rothman,

2009). The protein complexin act as the cofactor for vesicular fusion. The complexin contains

two short a-helices with flexible sequences, one of which is bound to the SNARE complex.

Before synaptic vesicle exocytosis, synaptotagmin interact with SNARE complexes by Ca2+

-

independent manner and position itself for subsequent Ca2+

-sensing. During the arrival of an

action potential, there is an influx of Ca2+

into the terminal and which induces synaptotagmin to

bind with phospholipids. This in turn displaces some part of complexin and opens the fusion pore

to trigger the release of neurotransmitter (Sudhof, 2013) ( Fig : 4)

Figure 4: Model for the action of synaptotagmin and complexin in the SNARE–SM protein

cycle ( Sudhof, 2013).

1.2.4 Post synaptic terminal

Post synaptic terminals have a series of receptors to sense the release of

neurotransmitters and produce responses based on the type of neurotransmitter. The response

may be either excitatory or inhibitory. If the neurotransmitter depolarizes the post synaptic

membrane, it produce an excitatory postsynaptic potential (EPSP), if it hyperpolarize the post

synaptic membrane, it bring an inhibitory postsynaptic potential (IPSP) (Tortora and Derrickson,

2009). Generally the EPSP is produced by glutamatergic synapses in which glutamate is the

neurotransmitter, which is received by NMDA (N Methyl D Aspartate) receptor or AMPA (α-

amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor located on postsynaptic

terminals (Sobolevsky and Rosconi et al, 2009). IPSP is produced by GABAergic synapses in

which γ-Aminobutyric acid (GABA) as the neurotransmitter, which are received by GABA

receptor on the postsynaptic terminal (Sigel and Steinman, 2012). All the above mentioned

receptors are called as ionotropic receptors as they functions as channel protein on binging of

neurotransmitters. Apart from the ionotropic receptors, there are metabotropic receptors which

sense neurotransmitter release but functions through G-protein coupled pathway for cellular

response (Tortora and Derrickson, 2009).

1.2.4a. NMDA receptor

The NMDA receptor belongs to a family of ionotropic glutamate receptors. This

receptor plays an important role in brain plasticity, especially in learning and memory and is one

of the most functionally regulated receptors in brain. The NMDA receptor currents increases

within 10–50 ms and which is much slower than those of non-NMDA receptors (0.2–0.4ms).

Likewise, NMDA receptors deactivate with a slower time rate 50–500 ms (Zito and Scheuss,

2009).

Structural organization of NMDA receptor-

NMDARs comprise three subtypes: NR1, NR2 and NR3. There are eight splice

variants for NR 1 (GluN1- 1a to GluN1-4a, and GluN1-1b to GluN1- 4b) , produced by

alternative splicing of exons 5, 21 and 22 ( Laurie and Seeburg, 1994; Bottai et al,1998 ;

Sugihara et al, 1992). There are four different NR2 subunits (GluN2 A-D), and two NR 3

subunits also ( GluN3 A-B), all of which are encoded by six separate genes. Functional

NMDA receptors require heteromeric complexes, with assembly of two GluN1 subunits

together with two GluN2 subunits or NR 3 (Monyer et al, 1992).

All the NMDA receptor isoforms has a conserved structural pattern that contain four

domains: the extracellular amino-terminal domain (ATD), the extracellular ligand-binding

domain (LBD), the trans membrane domain (TMD), and an intracellular carboxyl-terminal

domain (CTD) (Fig: 5).

Figure 5: Structure of glutamate receptor- linear representation of the subunit polypeptide

chain and schematic illustration of the subunit topology. (Sobolevsky and Rosconi et al,

2009).

The ATD (amino terminal domain) region of NMDA receptor have the binding

sites for divalent cations, such as Zn2+, negative allosteric modulators, (such as the

phenylethanolamine, ifenprodi), extracellular proteins (such as N-cadherin) and neuronal

pentraxins (NARP and NP1) (Traynelis and Wollmuth et al, 2010). The LBD (ligand binding

domain) of GluN1 and GluN3 subunits, act as the glycine binding sites and while in GluN2

subunits this domain act as the glutamate binding sites (Furukawa et al, 2005). The core ion

channel is formed by trans membrane helices M1, M3, and M4 from each of the four

subunits. The intracellular CTD (carboxy terminal domain) encodes short docking motifs,

mainly involve in membrane targeting, post-translational modifications, stabilization and

targeting for degradation (Traynelis and Wollmuth et al, 2010).

Function of NMDA receptor-

NMDA receptors have a critical role in brain function especially in encoding

memory. These receptors allows strengthening of synapses, through long-term potentiation

(LTP), and the weakening of synapses, through long-term depression (LTD), which are

proposed to be the cellular mechanisms involved in learning and memory (Luscher and

Malenka, 2012).

At the resting membrane potential, NMDA receptors are functionally blocked by

magnesium ions. Upon depolarization, the Mg2+ block is relieved; the receptor channel allows

influx of Na+ and Ca2+ ions and efflux of K+ ions (Luscher and Malenka, 2012). Apart from

removal of Mg2 block the receptor require binding of both glutamate and on the subunits:

GluN1/GluN3 provides the glycine or D-serine binding site and GluN2 provide the glutamate

binding site (Furukawa et al, 2005). These two interactions are required for maximum activation

of the receptor. The entry of calcium ions into the post synapse via the NMDA receptor, often

coupled the electrical synaptic activity, initiates biochemical signaling within the cell via

activation of Ca2+-dependent enzymes and downstream signaling pathways. This leads to the

activation of cyclic adenosine monophosphate response element binding protein (CREB), a

transcription factor, in turn allowing activation of genes essential for neuronal growth, such as

brain derived neurotrophic factor (BDNF). By this way long-term changes in synaptic strength

and other cellular modifications including alterations in synaptic structure or connectivity take

place. (Scheetz et al, 1994). In both LTP and LTD elevation of intracellular Ca2+ level occurs

through NMDA receptors. Modest increase in the post synaptic Ca2+ level triggers LTD and

stronger activation of NMDAR leads in large increase in Ca2+ level which triggers LTP.

Interestingly, it’s reported that during LTP more AMPA receptors are expressed in the synapse

and during LTD these receptor levels are low at the synapse (Luscher and Malenka, 2012).

Figure 6: Representation of magnesium block at resting membrane potential and the

removal of magnesium Upon depolarization and the conductance of ions ( Zito and

Scheuss, 2009).

1.2.5. PC12 cells as a neuronal model and the effect of NGF

PC12 cells line was derived from a solid pheochromocytoma tumor of adrenal

medulla from New England Deaconess Hospital strain white rats, which is responsive to nerve

growth factor ( NGF). The adrenal medulla consists of chromaffin cells and which has the

embryonic origin from neural crest cells. It has been using as a common model for neuronal

differentiation in vitro. Upon treating with NGF the cells stop division and differentiated to

neuron with elaborated branching and processes (Greene and Tischler, 1976). PC12 cells are

small (6 -14 μm in diameter), circular, releases catecholamine (dopamine as the major) and

acetyl choline, grows in serum supplemented media, with a doubling time of between 48 and 96

hr (Fujita et al, 1989).

NGF signaling in PC12 cells is mediated through the receptor tyrosine kinase

(RTK), TrkA which is responsible for differentiation. NGF was found to bind with Trk, resulting

in TrkA dimerization and trans phosphorylation. The phosphorylated Trk interacts with its target

proteins, PLCγ1 and SHC. PLCγ1 releases the second messenger molecules such as

diacylglycerol, which stimulate protein kinase C and increase intracellular calcium thus induce

further down-steam changes (Mischel et al, 2002). SHC on the otherhand activates ras cascade.

The activated ras binds to the serine- threonine kinase raf which in turn phosphorylates and

activate mek, a threonine and tyrosine kinase. It is found that the activities of both raf and mek

plays a central role for PC12 cell differentiation (Sun et al, 2006) Trk signaling pathway finally

regulates both CRE-binding protein (CREB) and CREB-binding protein resulting in gene

regulation (Vaudry et al, 2002).

Figure 7: Signaling pathways for PC12 cell differentiation by NGF and PACAP (Vaudry et

al , 2002).

1.3 Hypodissertation

In the nervous system, neurons form functional network for information exchange.

Based on the signals it receives, neurons either eliminate old connections, forms new

connections or stabilizes the previous connections. For studying these synaptic changes there is

an essential need for a simple model system, which forms functional connections within a short

period of time. Since synapses are the functional center for the neuronal network, it is critical to

understand how these connections are established and how the partner selections are set in. For

this behavior of synaptic plasticity, both post-synaptic and pre-synaptic proteins play a critical

role. For example NMDA receptor, a post synaptic protein, NR1 expression begins early on

embryonic 14 day (E14) then increases around the third postnatal week, and then declines on

slightly to adult levels. Different isoforms of synaptotagmin, a presynaptic protein, also express

in the biological system. The synaptotagmin also found to be increased during the development

and persist in the adulthood. However, the pattern of NMDA receptor and synaptotagmin

localization during early neuronal development, ie from undifferentiated level to different stages

of differentiated level largely remains unclear. PC 12 is an appropriate model to study changes

in the neuronal development, which upon treatment with NGF converts to neuronal like

structure.

The present study is based on the hypothesis that, a) Whether PC 12 cells can

develop into a functional neuronal network, and if yes, how efficient the development of

network? b) Whether the expression profile and localization of NMDA receptor and

synaptotagmin 1 changes as functional neuronal network establishes.

1.4 Objectives

Develop the cellular network formation of PC12 cells and measure the time kinetics.

To analyze the complexity of neuron on differentiation by Sholl analysis.

To visualize the formation of neuronal network by live cell imaging.

To compare the distribution and localization of NMDA receptor (NR1) and Syt1 in

differentiating PC12 cells using cell fixation and immunocytochemistry.

CHAPTER 2 - MATERIALS AND METHODS

2.1 Maintenance and Sprouting of PC12 Cells

DMEM F12 was purchased from Himedia Laboratories, India, Horse serum and Fetal

bovine serums were purchased from PAN biotech, Germany. Poly L lysinehydrobromide,

Penicillin G and nerve growth factor – 7s were purchased from Sigma Aldrich, Streptomycin

Sulfate was purchased from GIBCO, Germany. Sodium bi Carbonate was purchased from Sisco

Research Laboratories, India. Forma Direct heat CO2 incubator was from Thermoscientific. All

Optical Images were captured using Olympus IX51 microscope operated by the software

Application NIS Elements - Advanced Research supplied by NIKON. Polystyrene cell culture

plates and T25 flasks were from Nunclon, Denmark.

2.1.1 Poly L lysine coating

One ml of 0.1mg/ml solution of Poly L Lysine hydro bromide was added to each T-25

polystyrene flask (500μl Poly L Lysine hydro bromide solution for 60mm dish). The flasks/

dishes were then incubated for 15 minutes at 37°C. The solution was removed and the plates

were allowed to dry at room temperature at least for 2 hours before seeding the cells.

2.1.2 Cell culture maintenance

PC12 cells were maintained in DMEM F12 Media supplemented with 10%, Horse

Serum and 5% Fetal Bovine Serum 100 IU/ml Penicillin and 100μg/ml streptomycin in T-25

flask, at pH 7.4, under humidified atmosphere at 5%CO2 concentration and 37°C in CO2

incubator. Media was replenished in every third day.

2.1.3 Passaging of cells

On attainment of 70 to 80% confluence, the cells were trypsinised by the following

protocol. Media was removed from the T25 flask; the cells were washed with Phosphate

Buffered Saline (PBS). PBS was removed and added 800μL Trypsin/EDTA mix. The flask was

incubated at 37°C for 4 minutes. 800μL of DMEM F12 complete media was added after the

incubation time. The trypsinised cells were collected in a microfuge tube and centrifuged at

700rcf for 5 minutes at room temperature. The supernatant was discarded and the pellet was re-

suspended in new DMEM F12 complete media, counted the cell number and plated into new

PLL coated T25 flask at a density of about 5x105 cells per flask (1x105 cells per 60mm dish).

2.1.4 Cell counting

10μl of cell suspension was added to each side of the Newbauer counting chamber and

number of cells in 5 large squares was counted manually under microscope (in 10 X). Average

cell number was taken and number of cells per ml was calculated using the following equation.

Number of cells per ml = Average cell number x 10 x 103

2.1.5. Differentiation of PC 12 cells

PC12 cells were seeded at a density of 70,000 cells per 35 mm Poly L Lysine coated

polystyrene petridish in DMEM F12 medium, supplemented with 10% Horse Serum and 5%

Fetal Bovine Serum, 100 IU/ml Penicillin and 100μg/ml streptomycin pH 7.4. After 24 hours the

cells were attached and new medium: DMEM F12 supplemented with 1% fetal bovine serum,

200ng/ml nerve growth factor-7s and 50mM potassium chloride, at pH 7.4 was added. Media

was replenished every alternate day to ensure constant supply of the nerve growth factor and

nutrients and the cells were allowed to differentiate for six days. 10 Images were taken using

Olympus IX51 microscope in 20X magnification and used for further analysis.

2.1.6. Neurite Length Measurement

The length of the neurites was measured by tracing the each neurite using NIH Image J

free hand selection tool, and measuring the length of the tracing using ROI manager

2.1.7. Sholl Analysis

One representative cell was selected from each day of NGF treatment, and traced the

neurite using Fiji Plugin Simple neurite tracer ( Longair et al, 2011). Sholl analysis was

performed by image J software. Parameters like Critical Value (CV), Critical Radius (CR),

maximum radius were obtained. Schoenen’s Ramification Index (SRI) was calculated by the

following formulae.

SRI = Critical Value / Number of primary neuritis

2.1.8 Live cell imaging

Observed the neuronal network formation of differentiated PC 12 cells from day 2 to 6,

using JuLi smart fluorescent Cell viewer. 288 images were taken at 10 minute interval for 48

hours (10X magnification). The images were viewed in time lapse using JuLi software.

2.2 Fixation and Immunocytochemistry

Fetal bovine serums were purchased from PAN biotech, Germany. NR1 primary antibody was

purchased from Pierce Biotechnology. Syt1 primary antibody,FITC conjugated goat anti rabbit

secondary antibody and Hoechest stain were purchased from Sigma

From NGF treated PC12 cells (for day 0, 2, 4 & 6), media was removed completely ,

washed with PBS and fixed using 4% paraformaldehyde for 1 hour at room temperature. After

washing the cells in PBS, cells were permeabilized by 0.2% triton X 100 for 10 minute at room

temperature (in case of Syt1 only). After permeabilization, cells washed with PBS then blocked

in 5% FBS for 30 minute at RT, followed by probing with polyclonal rabbit Syt1 antibody

(1:100 dilution), and polyclonal rabbit NR1 antibody ( 1: 200) for each sample of NGF treated

cells ( for day 0, 2, 4 & 6) and incubated overnight at 4°C. After giving PBS wash added

secondary antibody (FITC conjugated goat anti rabbit secondary antibody -1: 100) and incubated

for 1.30 hour at room temperature. After giving PBS wash added Hoechest stain (1:1000) for 10

minute at room temperature, removed stain added PBS and taken images using Olympus IX51

microscope in 40X magnification.

CHAPTER 3- RESULT AND DISCUSSION

3.1 SPROUTING OF PC 12 CELLS.

The PC12 cells were differentiated by the treatment of nerve growth factor (NGF) and

followed up to 6th

day. Neuritic sprouting was observed with 24 hours of NGF treatment and by

6th

day extensive interconnecting neurites were observed (Fig: 8).

Figure 8: Neurite sprouting of PC12 cells after NGF addition on (A) day 0, (B) day 1, (C)

day 2, (D) day 3, (E) day 4, (F) day 5 and (F) day 6.

Neurite lengths were measured using image J software. A total of 100 neurites were measured

for the analysis. The result shows that, there is a progressive increase in the length of neurite

after NGF treatment, which reached the maximum on the 6th

day (Fig: 9).

Figure 9: Comparison of average neurite length on each day after NGF treatment

Sholl analysis was carried out to quantitatively measure the axonal dendritic population

among the neurites. A single neuron from each day after NGF treatment was traced using Fiji

plug-in simple neurite tracer software. Certain parameters like critical value, critical radius,

Sheonen’s Ramification index were calculated in the analysis. Critical value from the linear

profile represents maximum value of intersections. It reflects the highest number of processes or

branches, representing the complexity of dendrites and axons as the cell develop into a neuron.

Critical radius is the radius at which critical value occurs, which represents the maximum length

of dendrites. Shoenen’s Ramification index is the ratio between critical value and number of

primary branches directly arising from the soma, which measures the sub-branching within the

dendrites. Maximum radius is the measure of axonal length. Linear Sholl profile was taken for

analyzing neuronal complexity (Fig: 10-15).

Figure 10: Images of differentiated PC12 cells and linear profile of Sholl analysis (A) day 1

after NGF treatment (B) traces (C) merged image (D) linear Sholl profile.

Figure 11: Images of differentiated PC12 cells and linear profile of Sholl analysis (A) day 2

after NGF treatment (B) traces (C) merged image (D) linear Sholl profile.

Figure 12: Images of differentiated PC12 cells and linear profile of Sholl analysis (A) day 3

after NGF treatment (B) traces (C) merged image (D) linear Sholl profile.

Figure 13: Images of differentiated PC12 cells and linear profile of Sholl analysis (A) day 4

after NGF treatment (B) traces (C) merged image (D) linear Sholl profile.

Figure 14: Images of differentiated PC12 cells and linear profile of Sholl analysis (A) day 5

after NGF treatment (B) traces (C) merged image (D) linear Sholl profile.

Figure 15: Images of differentiated PC12 cells and linear profile of Sholl analysis(A) day 6

after NGF treatment (B) traces (C) merged image (D)

linear Sholl profile

The result shows that, though the neurite length was showing a linear pattern

(Fig:9),the branching pattern (critical value) was quiescent till 4th

day, after the initial sprouting

on day 1, till day 4, before showing a gradual increase in number of branching on day 5 and day

6 (Fig:16). The Ramification index (Fig:18) also follows a similar pattern, suggesting that the

dentitic branching started happening on day 5th

and 6th

. This is a strong indication that the

neurons are making stable connections as the dendritic filapodia allows the development of

dendritic spines (Grutzendler et al, 2002) from 5th

day onwards. Critical radius and Maximum

radius, which represents neurite length and axonal length respectively, shows a linear pattern of

development as expected (Fig: 17 and 19). With this analysis I could establish that the PC12

cells could make a stable neuronal network after Day 5 of differentiation.

Figure 16: Change in Critical value after NGF treatment (A) from day 1 to day 6 (B)

Percentage of Critical value change on each day compared to the value of day 6.

Figure 17: Change in Critical radius after NGF treatment (A) from day 1 to day 6 (B)

Percentage of Critical radius change on each day compared to the value of day 6.

Figure 18: Change in Shoenen’s Ramification index after NGF treatment (A) from day 1 to day 6

(B) Percentage of Shoenen’s Ramification index change on each compared to the value of day 6.

Figure 19: Change in Maximum radius after NGF treatment (A) from day 1 to day 6 (B)

Percentage of maximum radius change on each day compared to the value of day 63.2

The results of live cell imaging show that on early stages on neuronal development (by

the 2nd

to 4th

day) neuron tries to select their appropriate partners. The data suggest the synapse

formation is highly specific, as early random connections failed to establish (see Fig: 20). On

later stages (by the 4th

and 6th

day) most of the synaptic connections were stabilized (Fig: 21).

Figure 20: Formation of neuronal network on PC12 cells by 2nd

to 4th

day. Arrowhead in

the images show that a contact between neurites (A) was retracted within 10 minutes (B).

Further retraction was observed at 30 minutes after the formation of initial contact (C).

However a new branch formation was initiated by the neurite (D) which establishes a new

contact within 40 minutes (E).

Figure 21: Formation of neuronal network on PC12 cells by 4th

to 6th

day. Arrowhead in

the images shows that (A) a synapse formation by 5th days after NGF treatment. The

synapse showed no major changes even after 20 minutes (B), 1 hour (C) and 2.10 hour (D)

follow up.

3.2 IMMUNOCYTOCHEMISTRY

I have taken two candidate proteins to predict active network formation between PC12

cells, Syt1 (a synaptic vesicle protein at the pre synaptic terminal) and NR1 (an NMDA receptor

at the postsynaptic terminal). Both these proteins are critical in development of stable synaptic

connection. To analyze the change in the localization and distribution pattern of these proteins,

immunocytochemistry was performed on both undifferentiated and differentiated PC12 cells

(2nd

, 4th

and 6th

days of NGF treatment). Synpatotagmin expression was observed in PC12 cells

and it showed a punctuate distribution across selected neurites (Fig : 23 and see insert in Fig:

25), presumably axons. Synaptic vesicle migration is critical for axonal growth and it is also

known that NGF treatment induces extensive vesicular formation (de Carrisoza et al, 2010).

Axonal growth and active zone formation requires synaptic vesicle fusion; this helps in targeting

synapse specific protein to the plasma membrane (Ziv and Garner, 2004).

Our results showed that NR1 also expressed in both differentiated and undifferentiated

PC12 cells (Fig : 22). However the NR1 expression showed dramatic change targeting to

dendritic spine in Day 6 (see insert in Fig :24) suggesting there is specific targeting of NR1 to

active dentitic spines as neuronal network establishes. From the immunocytochemistry data we

could infer that it is the localization of NR1 is varying in the cells. The results suggest that PC12

cells are forming a functional connection within few days of network formation. This possibility

opens it as a very good model system to study the early stages of synaptic selection. Since the

cell has already gives an advantage to study zero time points of neuronal development, its ability

to form function network, could be exploited to understand nervous system development as well

as neurodegenerative diseases.

Figure 22: Immunocytochemistry images of NGF treated PC12 cells for NR1 on day 0

(A-C), day 2(D-F), day 4 (G-I) and day 6 ( J-I).

Day 0

Day 2

Day 4

Day 6

Fluorescence Nuclear staining Merged

Figure 23: Immunocytochemistry images of NGF treated PC12 cells for Syt1 on day 0 (A-

C), day 2(D-F), day 4 (G-I) and day 6 ( J-I).

Fluorescence Nuclear staining Merged

Day 0

Day 4

Day 6

Day 2

Figure 24: Immunocytochemistry images of NGF treated PC12 cells for NR1 on day 6. (A)

Fluorescence image (B) Phase image. Inserts show the localization of NR1 to the dendritic

spine.

A

B

A

Figure 25: Immunocytochemistry images of NGF treated PC12 cells for Syt1 on day 6. (A)

Fluorescence image (B) Phase image. Inserts show the localization of Syt1 to the axonal

terminal.

B

A

CHAPTER 4- SUMMARY AND CONCLUSION

In the nervous system, as the neuron gets differentiated, it forms functional

connections. During neuronal differentiation the synaptic proteins begin to target to their

respective sites. NMDA receptor is a post synaptic membrane protein involved in impulse

transmission, synaptic plasticity, learning and memory. Synaptotagmin, expressing on

presynapse has involved in neurotransmitter release and synaptic vesicle endocytotic pathway.

Their expression profile are expected to change as the neuronal cell differentiate and form

functional connections.

The expression pattern of NMDA receptor NR1 and synaptotagmin1 on early neuronal

development remain unclear. Previous reports show conflicting results on NR1 expression during

neuronal differentiation in PC12 cells. In the present study, it has observed that the complexity of

neuron increased after differentiation. The results suggest the sprouting neurites are forming

functional connections, with increased neurite length and branching density. The PC12 cells

could make an extensive neuronal network after Day 5 of differentiation which indicate that the

neurons are making stable connections as the dendritic filopodia allows the development of

dendritic spines.

The pattern of distribution of post synaptic protein NR1 and presynaptic protein

synaptotagmin also shows changes as the network gets established. NR1 was observed on both

undifferentiated as well as differentiated PC12 cells. As the neurite length increases, on each

day of differentiation, NR1 and synaptotagmin were found to be targeted to their functional sites.

NR1 was dramatically targeted to the post synaptic terminal. Synaptotagmin was found to

targeted to the axonal terminal. The data suggest that PC12 cells can be used to develop a

functional neuronal network to study a) how synapses are formed, b) which molecular pathways

attracts dendritic and axonal movements and c) how network behave after an injury. The major

advantage of the model is its easiness to develop a network within few days.

FUTURE PROSPECTS

In the present study it was observed that the expression of presynaptic and postsynaptic

markers is targeted to its functional positions. However it is unclear whether these molecules

play any role in partner selection and network formation. To know the role of these proteins on

differentiation, siRNA knockdown approach might give direct indication whether they have a

primary role in neuritic interaction and partner selection. Besides, a series of experiments using

patch clamp would provide direct evidence for functional connections within PC12 network.

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APPENDIX

Reagents and Buffers

DMEM F12 complete media ( for 50 ml)

DMEM F12 1: 1 solution 42.2 mL

Fetal bovine serum 5.0 mL

Horse serum 2.5 mL

Penicillin solution 50 μL

Streptomycin Solution 50 μL

DMEM F12 differentiating media ( for 50 ml)

DMEM F12 1: 1 solution 49.4 mL

Fetal bovine serum 0.5 mL

Penicillin solution 50 μL

Streptomycin Solution 50 μL

DMEM F12 1:1 mixture was made by dissolving 15.7 g in 1L of sterile distilled water.

Penicillin stock solution (100 IU/mL) was made by dissolving 30.165 mg of penicillin in 500 μl

of sterile distilled water and stored at 4°C streptomycin stock solution(100mg/ml) was prepared

by dissolving 50 mg of streptomycin in 500 μl of Sterile distilled water and stored at 4°C.

Phosphate buffered saline (PBS)

137mM NaCl

2.7mM KCl

10mM Na2HPO4

2mM KH2PO4

Dissolve 8g NaCl, 0.2g KCl, 1.44 Na2HPO4 and 0.24g KH2PO4 in 800mL of distilled

water. Adjust pH to 7.4 with HCl. Add H20 to 1 litre. Sterilize by autoclaving for 20 minutes at

15psi. Store at 4oC

Nerve Growth Factor - 7s stock

Nerve Growth Factor – 7s 1mg

DMEM F12 Differentiating media 1ml

Stored at -20°C in 10 μl aliquots to avoid repeated freeze thawing.

100x poly L Lysine Stock Solution

Poly L Lysine hydrobromide 10 mg

Sterile Distilled water 10 ml

Stored at -20°C