expression profile of nmdar and synaptotagmin in early...
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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