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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Microtubules: Role in axon and dendrites formation

Polarized cells such as neurons have two distinct domains i.e. molecular and functional domains which have been emerged from the cell body. In neurons, axon and dendrites are two distinct functional units emerged from the cell body by the process called neuronal polarity. Axons are thin, single and long structure which will transmit the signals to the target or synaptic partner. Whereas dendrites are shorter, multiple structures which has the ability to receive signals from different regions of cells or axons.

Before getting with role of microtubules in axon and dendrite formation, we need to look into the neuronal polarity part which is already been explained in one of my post. I.e. a hippocampal neuron polarize in the presence of insulin like growth factor (IGF-I) without any cell- cell interaction or extracellular matrix. Now these polarized cells exhibit the molecular features that distinguish axon and dendrites.

So in neuronal polarity, these are the list of protein involved:

1) Kinases

2) Phosphatase  

3) Small GTPase

4) Scaffolding proteins

Now what microtubule has to do with neuronal polarity?

Actually if polarity is lost, then it could be due to some modification in microtubule organization and dynamics. Hence microtubule has a very important role in neuronal polarity.

When do we say that neuronal polarization takes place?

It happens to place when there is local microtubule assembly and stabilization in one of the neurite. Change in the dynamics of the microtubule will lead to alteration in axon and dendrite specification.

Facts about microtubules:

Hollow cylindrical, covalent cytoskeleton polymers (24 nm diameter)

Made of tubulin proteins (α tubulin and β tubulin)

For polymerization Tubulin dimers bound to GTP

For depolymerization Tubulin dimers bound to GDP

α tubulin and β tubulin forms dimer

γ tubulin are attached at the minus end by nucleation. They form a ring at the minus which act as a cap to prevent further addition of any tubulin at the site.

    Microtubule structure

Two ends of microtubules: plus end (+) and minus end (-)

At Plus end (+), dimers with GTP are assembled and GTP bound to β tubulin gets hydrolyzed to GDP through inter dimer contacts. Growth and shrinkage of microtubule takes at this end.

At minus end (-), dimers gets dissociated and polymerization at this end is prevented by cap formed by γ-tubulin ring complex. Basically, minus ends are anchored to the centrosome or may be found as free ends in some cells.

Function: cell motility, mitosis, intracellular transport, secretion, maintenance of cell shape and cell polarization. 

Cycle of microtubule growth and shrinkage

Microtubule has the ability to undergo the cycle of growth and disassembly (as shown in the fig). They never reach the steady state length, but they exist in either polymerization or depolymerization state.  

There are two things happen in this cycle. One is catastrophe where depolymerization of the tubulin dimers takes. And other is rescue where the dimers polymerize together for the growth. 

These two things depend on the binding of GTP at the E site (nucleotide exchangeable site) of the β tubulin. During polymerization, the E site of the β tubulin has to be bound with GTP so that α tubulin can come and bind to it. Polymerization dynamics of microtubules are important for their biological functions because they allow themselves to rapidly reorganize, differentiate spatially, temporally in accordance with cell context, and to generate pushing and pulling forces during polymerization and depolymerization. 

Microtubule Associated proteins (MAPs)

These are the protein which joins the two microtubules together and form a bundle. There are two MAPs which are present in the axon and dendrites:

a) Tau protein

b) MAP2

a) Tau protein

Microtubules in axon has uniform orientation with their plus ends facing the axon tip and it’s bundled up together by the protein called tau protein. It’s also present in the somato-dendritic compartment and axon region. It’s not having any essential role in axon growth and microtubule stability. This statement was actually studied in tau knock out mice where its neuron showed no significant difference with that of the wild type mice. Except in one case where the tau deficient mice showed reduction in packing density and number of microtubules in cerebellar parallel fibers, a small type of caliber axon.

But this protein is important in the case of neurite outgrowth as well as growth cone motility. Studies have been taken place in chick sensory neuron (dorsal root ganglion) where inactivation of tau protein leads to reduction in the number and length of neurite.  In the growth cone, inactivation of tau protein lead to 20% decrease in the lamellopodial size and there lamellopodial motility is affected.     

Tau protein plays a vital role in development of Alzheimer’s disease.  

b) MAP2

Microtubules in dendrites have multiple orientations with their plus ends facing either the cell body or the dendritic tips and the bundling protein here is the MAP2.  It has 3 or 4 microtubule binding domains at the COOH- terminal, and is involved in microtubule assembly and stabilization in dendrites. MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.

Fig 3: Developed Neuron: Microtubules in Axon and dendrites are shown by magnifying the parts. Tau in axonal microtubule and MAP2 in somatic dendritic compartment. organelles such golgi apparatus,  polyribosomes and Endoplasmic reticulum are localized in the dendritic region.(Nature Neuroscience review)

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Growth cone and its role in axonal guidance

Growth cone are specialized and highly motile cellular compartment at the tips of the growing axon which supports the growth of the axon by sensing the extracellular cues and transducing it to the cytoskeletons.

The structure consists of a central region filled with organelles and microtubules; whereas at the peripheral region, it has highly dynamic, actin rich region such as Lamellipodia and Filopodia.

Lamellipodia are the broad veil like cellular protrusions that contain branched actin filaments. Filopodia are thin protrusions made out of unbranched and parallel F actin bundles. 

Fig 1: Shows that the central region of the structure is composed of densely concentrated organelles, whereas thin veil like Lamellipodia and spiky Filopodia is found at the peripheral region.  

This is large paused growth cone imaged through differential interference contrast microscope. 

A brief about growth cone and its structure is explained in the topic called Cytoskeleton Role in Neuronal polarity.

A growth cone helps the developing neuron to reach the particular target site or synaptic partner. Hence axonal growth cones used to navigate along the specific pathways in the response to the molecule guidance cues. In this article, it will be explained that how growth cone is influenced by extracellular cues inorder to select a particular pathway towards the target site.

The growth cone is highly concentrated with actin filaments rather than microtubule. Microtubules which are bundled together in the axon shaft will also extend into the central region of the growth cones, but the growth cone stalls it, thereby making it to form a prominent loop in the central region.

Fig 2: distribution of actin and microtubule in the growth cone. 

In the Filopodia region, the actin filaments will constantly undergo polymerization and depolymerization. At the barbed end there will be addition of G actin molecule, whereas the in the pointed end the G actin molecules are constantly removed, thereby leading to the movement and extension of Filopodia. The G-actin molecule is brought back from the peripheral region to the distal region by myosin (actin cargo carrying protein) and this process we term as retrograde movement. The tip of the Filopodia contains the receptors for the extracellular cues and in the Lamellipodia is concentrated with regulators of actin and microtubules which gets activated and deactivated based the signals obtained at the peripheral region of the growth cones. Combining these two properties, growth cone is said to be the compartment which has the ability to guide the neuron to reach its target site or synaptic partner. 

Fig 3: Pictorial representation of growth cone action towards the gradient. 

In the fig 3, it’s shown how the cytoskeletons modify which lead the growth cone to turn towards the highly concentrated gradient. The polymerization and depolymerization of the actin takes place simultaneously in different regions. For eg: In the stage 3, the filaments are stable and polymerize towards the attracting gradient region, whereas there happens to be depolymerization in the non gradient region of the Growth cone. 

Cytoskeletons	Stage 1	Stage 2	Stage 3	Stage 4
Actin	Balanced F actin dynamics across the GC	Increase in anticapping and severing, decrease in capping leading to polymerization	Cross linking of the filament leads to stable ribs.	Capping of the F actin filament will not allow the polymerization and finalize to stabilization.
Microtubules	Random MT exploration of P region	Polymerization happens with less catastrophe	Acetylation and detyrosination leads to stabilization of microtubule.	Bundling

  Table 1: Action of cytoskeletons in the growth cone under two gradient regions. 

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Axonal initial segment

Information flows along a neuron in one direction i.e. synapse to cell body, then to axon initial segment (AIS), from their its passed to Axon and then to terminal region. Actually, neurons will receive both excitatory and inhibitory synaptic inputs on their cell bodies and dendrites. The fire of the repeated synaptic inputs will be summated and bursted out as action potential which will be taken cared by AIS. Axonal initial segment (AIS) is made up of high densities of voltage gated Na⁺ and K⁺ channels which initiate and modulate action potential. In the myelinated axons of vertebrates, the propagation of action potential is rapid along the axon through the activated cluster of sodium channels at the node of Ranvier. The action potential reaches the axon terminal and helps in release of neurotransmitters to propagate the signal across the synaptic cleft to another cell. 

Fig 1: Show the sequence of information follow through the neuron from the synapses. The AIS can be seen soon after the beginning of the axon (green) which consists of node of Ranvier. Node of Ranvier is found at different regions of the axon which consists of cluster of sodium channels.  

Both AIS and node of Ranvier consists of ion channels, cell adhesion molecules, extracellular matrix molecules, proteins like kinases, accessory proteins and cytoskeleton scaffolds.

The axonal initial segment is enriched with sodium gated channels which facilitates a high sodium current density and a low action potential threshold.

The assembly of AIS is actually an intrinsic property of neuron where no extracellular or glial dependent cues are required. But the formation and localization of node of Ranvier is regulated by glial derived signals. 

Ankyrin G

A cytoskeleton scaffold protein which is a master organizer of membrane domains and subcellular polarity in many cell types. This protein is restricted to AIS and AIS targeting motif located in the cytoplasmic loop of the neuron which connects the domain II and III of sodium channels binding to AnkG.  Sodium channel and AnkG interaction is facilitated by Phosphorylation of AIS targeting motif by casein kinase II (CK2), which is enriched at the AIS and node of Ranvier.

Removal or silencing of AnkG protein will lead blocking the clustering of sodium channels at AIS. And also it will lead to blocking the subcellular polarization of potassium channels KCNQ2 and KCNQ3, the cell adhesion molecules neurofascin and neuronal cell adhesion molecules NrCAM, the AIS extracellular matrix and the cytoskeleton protein IV spectrin.

From this we can learn that AnkG functions as a scaffold to which all other AIS proteins are tethered directly or indirectly and consequently AnkG establishes the subcellular polarity of these molecules.

The voltage gated sodium channel Nav1.6 and Nav 1.2 are found in the distal and proximal AIS.  The Kv1 family is also found primarily in the distal AIS.   

Fig 2: shows the structure of AIS 

 Axonal initial segment is composed of the following thing.

1. Ion channels (Nav1.x, KCNQ2–KCNQ3 and Kv1.x)
2. Neuronal cell adhesion molecule (NrCAM)
3. Neurofascin 186 (NF186)
4. Disintegrin and metalloproteinase domain-containing protein 22 (ADAM22)
5. Transient axonal glycoprotein 1 (TAG1, also known as contactin 2)
6. CASPR 2
7. Extracellular matrix molecules (brevican and versican)
8. Cytoskeletal scaffolds (AnkG, "IV spectrin and postsynaptic density protein 93 (PSD93))

Protein with unknown role in AIS

1. Casein kinase II (CK2)
2. Phosphorylated nuclear factor-κB (pNFκB)
3. Phosphorylated inhibitor of κBα (pIκBα)

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