Role of Fibroblast growth factor (FGF) in neural stem cell growth

Fibroblast growth factor (FGF) constitutes a large family of polypeptide growth factors found in a variety of multicellular organisms, including invertebrates.

The function of FGFs is not restricted to cell growth. Instead, FGFs are involved in diverse cellular processes including chemotaxis, cell migration, differentiation, cell survival, and apoptosis. The common feature of FGFs is that they are structurally related and generally signal through receptor tyrosine kinases. FGFs play an important role in embryonic development in invertebrates and vertebrates.

The human FGF protein family consists of 22 members that share a high affinity for heparin as well as a high-sequence homology within a central core domain of 120 amino acids, which interacts with the FGFR.

Structure of a generic FGF protein contains a signal sequence and the conserved core region that contains receptor- and HSPG-binding sites. The main structural features of FGFRs including Ig domains, acidic box, heparin-binding domain, CAM-homology domain (CHD), transmembrane domain, and a split tyrosine kinase domain are illustrated with respective functions: CAM, Cell adhesion molecule; ECM, extracellular matrix; PKC, protein kinase C.

FGFR signal transduction

The signal transduction starts soon after the FGF binds to the Ig domain III. Binding of FGFs causes receptor dimerization and triggers tyrosine kinase activation leading to autophosphorylation of the intracellular domain. Tyrosine autophosphorylation controls the protein tyrosine kinase activity of the receptor but also serves as a mechanism for assembly and recruitment of signaling complexes. These phosphorylated tyrosines function as binding sites for Src homology 2 and phosphotyrosine binding domains of signaling proteins, resulting in their phosphorylation and activation. A subset of Src homology 2-containing proteins such as Src-kinase and phospholipase Cγ (PLCγ) possesses intrinsic catalytic activities, whereas others are adapter proteins. FGF signal transduction, as analyzed during early embryonic development, can proceed via three main pathways and i.e.

Ras/MAPK pathway

The most common pathway employed by FGFs is the MAPK pathway. This involves the lipid-anchored docking protein FRS2 (also called SNT1) that constitutively binds FGFR1 even without receptor activation. Several groups have demonstrated the importance of FRS2 in FGFR1-mediated signal transduction during embryonic development. After activation of the FGFR, tyrosine phosphorylated FRS2 functions as a site for coordinated assembly of a multiprotein complex activating and controlling the Ras-MAPK signaling cascade and the Phosphatidylinositol 3 (PI3)-kinase/Akt pathway. The FRS2 tyrosine phosphorylation sites are recognized and bound by the adapter protein Grb2 and the protein tyrosine phosphatase (PTP) Shp2. Grb2 forms a complex with the guanine nucleotide exchange factor Son of sevenless (SOS) via its SH3 domain. Translocation of this complex to the plasma membrane by binding to phosphorylated FRS2 allows SOS to activate Ras by GTP exchange due to its close proximity to membrane-bound Ras. Once in the active GTP-bound state, Ras interacts with several effector proteins, including Raf leading to the activation of the MAPK signaling cascade. This cascade leads to phosphorylation of target transcription factors, such as c-myc, AP1, and members of the Ets family of transcription factors.

PLCγ/Ca²⁺pathway

The PLCγ/Ca²⁺ pathway involves binding of PLCγ to phosphorylated tyrosine 766 of FGFR1.Upon activation, PLCγ hydrolyzes phosphatidylinositol-4,5-diphosphate to form two second messengers, inositol-1,4,5-triphosphate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas inositol-1, 4, 5-triphosphate stimulates the release of intracellular Ca²⁺. This cascade has been implicated in the FGF2-stimulated neurite outgrowth (48, 49) and in the caudalization of neural tissue by FGFR4 in Xenopus.

PI3 kinase/Akt pathway

The PI3 kinase/Akt pathway can be activated by three mechanisms after FGFR activation, and the phospholipids thereby generated regulate directly or indirectly the activity of target proteins such as Akt/PKB.

Among other processes, the PI3 kinase signaling branch is involved during Xenopus mesoderm induction acting in parallel to the Ras/MAPK pathway. Overexpression of a dominant negative form of the PI3 kinase-regulatory subunit p85 interferes with Xenopus mesoderm formation. Conversely, co expression of activated forms of MAPK and PI3 kinase leads to synergistic mesoderm induction. 

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Hedgehog protein – signaling pathway in vertebrate neural development

Hedgehog family of proteins is signaling proteins which control the cell growth, survival, fate and pattern (almost every aspect) of the vertebrate body plan.  The name was derived from the short and spiked phenotype of the cuticle of the Hh mutant Drosophila larvae.

Except in C.elegans, It is found in both vertebrates and invertebrates. In C.elegans, it has several proteins homologous to the Hh receptor Ptc.  

There are three subgroups in hedgehog protein family:

                    A.  Desert hedgehog (Dhh)

                    B.  Sonic hedgehog (Shh)

                    C.  Indian hedgehog (Ihh)

Both the vertebrates and invertebrates, hedgehog binds to the receptor patched (Ptc) and activates a signaling cascade which ultimately drives the activation of the zinc finger transcription factor (Ci in Drosophila and GLI-3 in mammals) leading to activation of specific target genes.

 A.      Desert hedgehog protein:

The expression of this protein is restricted to gonads only which includes sertoli cells of testis and granulose cells of ovaries. Its deficiency in mice has not shown any significant phenotypic changes, but in males it leads to infertile since there is absence of mature sperm. Dhh is required for organogenesis, Leydig cell formation and sex cord formation. In the absence of Dhh signaling, the size of the precursor Leydig cell population is unaffected but these cells have reduced expression of the transcription factor steroidogenic factor 1 (SF-1). This is the likely cause of impaired expression of steroidogenic enzymes and ultimately testosterone, which is required for virilisation and spermatogenesis.

B.      Sonic hedgehog protein:

Basically it’s a mammalian hedgehog signaling molecule which is expressed during the early vertebrate embryogenesis in midline tissues such as node, notochord and floor plate to control the patterning of the left –right and dorso ventral axes of the embryo. In the zone of polarizing activity (ZPA) of the limb bud, Shh is expressed and critically involved in patterning of the distal elements of the limbs. During organogenesis also, it is expressed in and affects the development of most epithelial tissues. In the absence of the Shh protein will lead to cyclopia, and defects in ventral neural tube, somite and foregut patterning. The protein is very important for hypothalamic and pituitary development.

C.      Indian hedgehog protein:

The expression of this protein is limited to number of tissues which includes primitive endoderm and prehypertrophic chondrocytes in the growth plates of bones. During embryogenesis its absence in the embryo has lead to its death due to poor development of yolk sac vasculature. 

Hedgehog signaling pathway:

A general outline of the hedgehog signaling pathway in the cell is shown. After the translation of hedgehog, it undergoes multiple processing steps that are required for generation and release of the active ligand from the producing cell.

Maturation of the protein (Hedgehog)

1.       The signaling molecule undergoes a self cleavage soon after the signal sequence is removed. The cleavage is catalyzed by its own C terminal domain that occurs between the conserved glycine and Cysteine residues.

2.       The peptide between the two residues is rearranged to form a thioester. Now, oxygen of the –OH group of cholesterol attacks the -CHO of the thioester. This lead to displacing of the sulfur and cleaving of the Hh protein into two parts:

 Ø   A C -terminal processing domain with no known signaling activity.

 Ø  An N- terminal Hh signaling domain (HhN) of approx 19 kDa that contains an ester linked cholesterol at its C terminus. This cholesterol modification will result in association of the HhN with the plasma membrane of the cell.

3.       A palmitic acid moiety which is required for the HhN activity is added to the N terminus of Hh by the acyl transferase skinny hedgehog, this process is called palmitylation.

Without the cholesterol modification, the protein can escape through the dispatched protein easily and can be palmitoylated later during the transport or at the receiving cell. Hence Cholesterol modification role is found to negligible or more study need to be done on it. But palmitylation role is found to be important for generating soluble multimeric Hh protein complexes and also for long range signaling in vertebrate.  

Signaling process: 

Finally the matured Hh protein binds to the membrane protein called Dispatched, which will let the protein to be secreted out of the cell. Loss of the dispatched protein will cause the accumulation of the Hh protein in the producing cells and failure of long range signaling occurs.

The concentration and transport of the Hh protein is controlled by the extracellular proteins such as you/Scube 2 (in the case of zebra fish) as well as proteins on the surface of cells situated between those producing and receiving hedgehog signals (for example, patched (PTC1) hedgehog interacting protein (HIP), exostosin (EXT1), CDO (interference hedgehog (IHOG) in Drosophila) and its relative BOC).

In the absence of the Hh protein, the patched receptor (Ptc) inhibits the Smo (GPCR smoothened) receptor present either on the cell surface or on a vesicle inside the cell. This inhibition will lead to the activation of GLI3R protein which indirectly inhibits the class I Transcription factors such as PAX6, PAX7.  Therefore Hh target genes are not transcribed.

But when the Hh protein is released by the secreting cells, it binds to the Ptc1 receptors leading to activation of the Smo receptor. This inturn will inhibit the GLI3R formation and supports the formation of GLI2/3A from GLI2/3 with the help of protein like wimple, flexo, KIF3A and Rab23.

Finally GLI2/3A will activate the transcription factor (class I and II) and also GLI1 leading to transcription of Hh targeted genes. 

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Neuronal Polarity Introduction

The formation of single axon and multiple dendrites in a neural cell through distribution of specific functions to discrete cellular domains.

The neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Hence they start as a round neuronal spheres and gradually adopting a complex morphology by forming one long axon and several shorted dendrites to eventually connect to target cell or other neurons via synapses.  The central importance for the axon formation and the neuronal polarity is the specialized, highly motile cellular compartment at the tips of the growing axons called growth cone. 

Different stages of neuronal polarity 

Stage 1: Neuronal development starts with round spheres that spread a lamellipodium around the cell body

Stage 2: Then transform into cells containing several neuritis, which are decorated with dynamic growth cones at their tips.

Stage 3: Neurites at this early developmental stage show characteristic alternations of growth and retraction. The major polarity event is when one of these equally long neurites starts to grow rapidly to become the axon.

Stage 4: The next step is the morphological development of the remaining short neurites into dendrites.

Stage 5-6: Here Functional polarization of axons and dendrites, including synapse formation happens and dendritic spines are formed at later stages.

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Cytoskeleton Role in Neuronal polarity

Cytoskeleton plays a vital role in both establishing as well as maintaining polarity in neurons. Basically actin filaments and microtubules have the functional properties which make them uniquely suited to determine and regulate polarity in neurons as well as in other polarizing cells.

Two basic characteristic features of Actin filament and microtubules in polarity:

1.        The Microtubules rapidly convert the molecular signals into structural changes modulating cell shape.
2.           They possess an intrinsic polarity.

Growth cones support the growth by sensing environmental cues and transducing those signals to the cytoskeletons.

The structure (axonal growth cone) is basically composed of a central region filled with organelles and microtubules, whereas the pheripheral region is highly dynamic, actin rich area containing lamellipodia and filopodia.

it’s shown here that the barbed ends of the actin filament which is oriented towards the rim and latter pointed ends are towards the base of the growth cone. We can find that G-actin subunits are continuously incorporated into the barbed end while the other pointed end is found to be dissociated which is basically resulting in a “treadmilling” of F actin and regulation of growth cone dynamics.

In the initial establishment of the polarity, rearrangement of the actin cytoskeleton and microtubules is very important. There is an enhanced growth cone dynamics shown by the future axon before the occurrence of morphological polarization (circled in black).  Even the future dendrites are not growing properly at that stage, therefore it have a static growth cone with a rigid actin cytoskeleton (circled in red). If we pharmacologically depolymerase the actin cytoskeleton, then the non growing dendrites will be transformed into growing axons. It suggests that the actin filaments of the future dendritic growth cones form a barrier for the protrusion of microtubules, whereas the axonal growth cone contains an actin structure permissive for the microtubule protrusion.

Microtubules also actively participate in the neuronal polarization and they are more stable in the axonal region compared to the minor neurites. The stabilization of microtubules is far more sufficient to induce axon formation.      

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Neuronal polarity regulation

The hippocampal neurons form a spikes and small proturusion veils at stage 1. This is stage where the neuron stem cell breaks out from the cell cycle.

Then the truncated protrusions develop into a several neurites at stage 2.  

At stage 3, All the neurites are roughly equal in length, in which only one neurite will start to break the initial morphology symmetry and grows at rapid rate and thus they eshtablish polarity.

The extracellular cues can guide which neurite to grow as axon. The extracellular cues are highly concentrated over the stage 2 immature neurites. Well the guidance happens through the help of downstream signalling molecules present within the neurites. PI 3 kinase and its lipid products such as PIP3 determines and maintains the internal polarity.  PI 3 kinase activity is localized at the tip of the newly specificed growing axon and its activated when its get the signal from the extracellular cues.

The Signalling cascades helps the centrosome to position at one of the neurite and these centrosome position is responsible for the growth of a axon, because it guides the microtubules to move in one particular neurite. If a neuron is having multiple centrosomes, then it will lead to formation of multiple axons. From this its well understood that centrosome is indirectly responsible for the axon formation, since its controlled by PI 3 kinase, cdc 42 and other regulatory molecules.

Soon after the axon starts it rapid growth action, the other neurites enlongates and acquire the charactersitics of dendrites which is at stage 4.

Finally the neurons form the synaptic contacts and eshtablish a neuronal network at stage 5.

So at stage 2, There happens to be two kinds of feedback signals which is very balanced. They are positive and negative feedback signals. In this negative regulations, the actin dynamics decrease and microtubule catastrophe occurs, whereas in positive regulation, actin dynamics increases and microtubule assembly occurs. Hence the neurites are found to be of same length and it wont grow unless some extracellular cues triggers the downstream regulating molecules. When balance is broken, then the axon growth occurs, where the postive feedback plays a major role than the negative feedback loop.

PIP3, Akt and GSK 3β signaling cascade determines the fate of the axon growth. When the receptor gets activated or triggered by some extracellular cues like netrins or BDNF, then it’s the Ras, which is a G protein activates the PI-3 kinase. The PI-3 kinase converts the lipid membrane molecule PIP2 to PIP3 which activates the PDK 1 (Phosphoinositide dependent kinase 1). The PDK 1 and other kinases such as integrin linked kinase phosphorylates the kinase Akt protein (PKB) at threonine 308 and serine 473. The phosphorylated Akt protein will inactivate the GSK 3β by Phosphorylation.

GSK 3β will activate the regulator proteins which is will be responsible for the microtubule catastrophe. Hence the activated GSK 3β or overexpression of GSK 3β will not allow the formation of axon, whereas knockout of GSK 3β gene have lead to formation of multiple axon. Therefore, the GSK 3β will be found inactive at the tips of the growing axon or growth cone.  

RAP 1B (ras related protein 1B), which is ras superfamily GTPase localize at the tips of the axon before the cdc 42 and PAR complex accumulates. It is said that the accumulation RAP 1 protein in one particular neurite is responsible for the axon specification and recruitment of other proteins for axonal growth.  The TrkA A receptor is activated by the extracellular cues, which will consequently recruits the GEFs of RAP 1 and adaptor protein (CRK) and then stimulates the RAP1.

Rho GTPase: Rac 1 and cdc 42 activation will lead to the elongation of the neurite, whereas in the case of Rho A activation will lead to the inhibition of the neurite outgrowth. Certain Rho GTPase effectors also influence in the neurite outgrowth such as P35, PAK, MRCK, N- WASP and IQGAP3. There are regulators of Rac 1: TIAM 1 and STEF (SIF and TIAM 1 like exchange factors), which regulates the upstream molecules of it. 

PAR complex: PAR 3 and PAR 6 with aPKC will form a complex with cdc42. The PAR complex will activate the Rac 1 protein through the TIAM 1 and STEF. 

This picture shows how the PAR complex gets localized. They are transported from the cell body to the growing axon by the motor protein kinesin 2.  

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PAR protein in axon specification and neuronal polarity

Par (for partition defective) proteins are evolutionary conserved proteins, which are involved in regulating axon specification and establishment of neuronal polarity.  These proteins are first discovered in the C.elegans, which has 6 par genes named as par1-6.

s. no	Par proteins	In C.elegans	Drosophila homolog	Mammalian homolog	Involvement in neuronal polarity?
1	Par1	Serine/Threonine kinase	Par 1	MARK kinase SAD kinase	Yes
2	Par2	Zinc finger and ATP binding motif (ubiquitination pathway)	Not conserved	Not conserved	unknown
3	Par3	Scaffold (PDZ domains)	Bazooka	mPar3 (also called as ASIP PHIP)	Yes
4	Par4	Serine/Threonine kinase	Par4	LKB1	Yes
5	Par5	14-3-3 protein	Par5	14-3-3 protein	Unknown
6	Par6	Scaffold	Par6	mPar6	Yes

Par3/Par6/aPKC complex in neuronal polarity

Par3/Par6/aPKC complex plays a crucial role in axon specification and polarity establishment in a cultured hippocampal neuron. These complexes (Par3/Par6) are selectively enriched at tip of the future axon, inorder to make the cell polarized. If the complex expression is inhibited, then the neurite differentiating to axon and dendrite does not take place. If the complex is over expressed, then multiple axon or dendrite is formed.

The par3 and par6 are scaffold proteins, where Par 3 interacts with Par6 and with Tiam1 (T-lymphoma invasion and metastasis 1) and GNEF (Guanine nucleotide exchange factor) for Rac. Par6 forms a stable complex with aPKC (protein kinase C) and contains a semi- cdc42/Rac interactive binding (CRIB) domain that specifically binds to the active GTP bound form of the small GTPases, Cdc42 and Rac1 (central regulators of actin cytoskeletal dynamics).    

It also regulation of these par complexes is responsible for the control in morphogenesis of dendritic spines. 

Cdc42 which is activated by the PIP3 will initiate the positive feedback loop by binding to the Par6 protein.  

The par3/par6/aPKC complex will then activate Rac GEF which has STEF/ Tiam1 protein bind to it. This activated Rac complex will activate the Rac, a small GTPase protein and finally triggers the PI3 kinase. PI3 kinase is the important enzyme in neuronal polarity and axonal specification because it converts the PIP2 to PIP3, a molecule responsible for activation of other proteins playing a vital role in the axon growth. 

The par 3/ par6 complex will also inhibit the GSK3β which is responsible for the inhibition of CRMP2, Tubulin, APC and microtubule associated proteins (MAPs). 

Definitions


Axon Specification

In establishing a polarized neuron, the initial event is the specification of a single axon where one of the neurite will grow into a axon.

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