Advanced Neurobiology

Astrocyte: Structure, Function and Disease

2013-09-05T18:11:00.000+05:30

Astrocytes are star shaped glial cells which are found associated with the neurons of the CNS and spinal cord.

They modulate the aspects related to neural network functions.
Express the same sets of ion channels as neurons, but in varying proportion.
Maintenance of K+ and water homeostasis in the CNS via coordinated action of Kirl.4 and aquaporin 4 channels.
Gap junctions form intercellular networks and help in sequestering K+ and glutamate.
Modulation of synaptic transmission.

1. Lamellar fibrous astrocytes

Found in white matter
Oriented parallel to the neuronal axons
Higher levels of GFAP
End feet envelops the node of Ranvier

2. Smooth radial astrocytes

Found in cerebellum equipped with a large repertoire of receptors which helps in sensing the activity of synapses.
High densities of glutamate transporters.
Cell body spans both gray and white matter.

3. Protoplasmic astrocytes

Found in gray matter of CNS.
At least one of their process is in contact with the blood vessels.
Low input resistance.
Very negative membrane potential.
Prominent glutamate uptake.
Voltage and time independent K currents.
End feet envelops the synapses.

GFAP – Glial fibrillary acidic protein: biomarker of Astrocytes.

Plasma membrane ion channels

1. Cx43 channels

It’s selective for second messengers (cAMP, IP3 and calcium), amino acids (Glutamate, aspartate and taurine), Nucleotide (ADP, ATP, CTP and NAD), Energy metabolites (glucose, glucose 6 phosphate and lactate), small peptides (glutathione) and RNA (24 mer).

Not permeable for large molecules such as nucleic acids, proteins and lipids.

2. Cx30 channels

S.No	Receptors	Transporters
1	Purine receptors	Glutamate transporter
2	mGlu receptors	Glucose transporter

Uptake of glutamate by glial specific transporters:

EAAT1 (Excitatory amino acid transporters) (in rodent it’s known as GLAST (glutamate aspartate transporter) and GLT1 (glutamate transporter 1)) and EAAT2.

Function of Astrocytes:

(1) Astrocyte functions include modulation of synaptic function via glutamate transporters, which convey glutamate from the synaptic cleft into the cell.

(2) Communication between astrocytes occurs via ATP release and binding to purine receptors on adjacent astrocytes. ATP binding results in phospholipase C activation, with subsequent downstream activation of inositol trisphosphate, resulting in calcium mobilization.

(3) Gap junctions contribute to an astrocyte syncytium for the exchange of small molecules and cell–cell communication.

(4) Metabolic functions include

The replenishment of neuronal glutamate via the glutamate glutamine cycle. (4)
The transport of glucose from the vasculature. (5)

(6) The regulation of blood flow is modulated by astrocyte end-feet apposing blood vessels, with vasodilation being mediated through release of vasoactive substances.

(7) Glutamate release might occur following elevations in intracellular calcium and the activation of other factors related to prostaglandins.

(8) Glutamate release through hemichannels can be induced in vitro through lowering of extracellular calcium.

(9) Glutamate binding to metabotropic glutamate receptors activates intracellular calcium, leading to the release of vasodilatory substances. Abbreviations: Gln, glutamine; Glu, glutamate; IP3, inositol trisphosphate; PLC, phospholipase C.

Extra points:

Glutamate released from neurons increases glucose trafficking in Astroglial networks by activating AMPA (a-amino-3-hydroxy-5-methyl-4-Isoxazole propionic acid) receptors.

Homotypic Cx30 and Cx43 channels and heterotypic Cx43 – Cx30 channels are voltage dependent.

Astroglial networks supply glucose and lactate to sustain hippocampal synaptic transmission and studies investigating the role of IP3 in hippocampal function suggested that this molecule can induce glutamate release from gap junction coupled astrocytes, triggering transient depolarization and epileptiform discharges in CA1 Pyramidal neurons.

Function related to neural formation:

Control of synapse formation and function

Regulating number, size and shape of dendritic spines in Hippocampal CA1 pyramidal neurons via EphA3-A4 receptors.

Adult neurogenesis

Instructing neuronal fate commitment and by promoting proliferation of adult neural stem cells. Moreover the effects of astrocytes are regionally specified: astrocytes from adult hippocampus retain the potential to promote neurogenesis, but astrocytes from adult spinal cord do not.

Astrocyte excitation happens in two main forms:

Generated by chemical signals in neuronal circuits(Neuron dependent excitation).

Occurs independently of neuronal input (spontaneous excitation).

Propagation of glutamatergic transmission via the astrocyte network.

Propagation of glutamate transmission through the astrocyte syncytium occurs through two prominent calcium-mediated mechanisms:

Gap junctions
Paracrine release of ATP

Activation of metabotropic glutamate receptors on astrocytes following neuronal release of glutamate results in the activation of an inositol trisphosphate (IP3) pathway, which induces calcium release from intracellular stores. This calcium can then be transferred to the adjacent astrocyte through connexin 43 (Cx43) gap junctions, thereby producing a calcium wave through an astrocyte syncytium. IP3 also activates ATP release through Cx43 hemichannels. This ATP release acts in a paracrine fashion, activating purine receptors on adjacent astrocytes. This activation results in IP3 production, more ATP release and intracellular calcium mobilization through a feed-forward mechanism.

Astrocytes association with Neurodegenerative Disease

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

2011-12-24T19:11:00.000+05:30

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.

Hedgehog protein – signaling pathway in vertebrate neural development

2011-12-03T21:21:00.000+05:30

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.

Microtubules: Role in axon and dendrites formation

2011-11-13T22:09:00.005+05:30

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)

Growth cone and its role in axonal guidance

2011-11-06T19:37:00.000+05:30

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.

Axonal initial segment

2011-11-03T15:00:00.000+05:30

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.

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

Protein with unknown role in AIS

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

Centrosome role in neuronal polarity

2011-11-01T18:58:00.000+05:30

Centrosome or MTOC (Microtubule organizing center) is a kind of organelle found in both plant and animal cell which guides the formation of microtubule in the cell. Microtubule array arises from the Centrosome and spreads all over the structure of the normal cell. But in the case of developing neuron, the Centrosome is localized towards one particular neurite and then the microtubules are guided to that neurite which further forms the axon. Centrosome positioning is an important phenomenon in the neuronal polarity because out of all the neurite; one specific axon has to be formed towards the target cell or neuron.

Centrosome positioning takes place at the stage 3 of neuronal polarity, where the Centrosome is found closer to one particular neurite which is said to be the future axon.

After the cell exits from the cell cycle, it forms neurites. And then certain extracellular cues activate the intracellular cues which take the responsibility of Centrosome positioning. Centrosome positioning is the beginning stage of axon specification.

Structure of Centrosome is made up of two barrel shaped centrioles and a cloud of pericentriolar material that surrounds them. Microtubules are nucleated from the pericentriolar material and form a radial array emanating away from the Centrosome. Nucleation from the Centrosome regulates key features of the microtubules within the array. First, all of the microtubules assemble with their plus ends away from the Centrosome, resulting in a microtubule array of uniform polarity orientation.

This fig shows that the difference between the nonneuronal and neuronal cell’s centrosome activity. The pluripotent precursor cell can give raise to either nonneuronal cell or neuronal cell. In the nonneuronal cell, a portion of the microtubules nucleated by the centrosome are captured by the leading edge of the cell. The motility of the leading edge pulls on the microtubules, and the attached centrosome reacts by relocating in the direction of cell movement. In the neuron, the microtubules are released, and the centrosome is not relocated. Nevertheless, the microtubules are translocated toward the leading edge, which coalesces into a growth cone. The cell body remains stationary and the microtubules translocate into the space between the cell body and the growth cone, which develops into the axon.

Microtubules destined for the axon are initiated at the centrosome and then released for translocation. Released microtubules are transported through the cytoplasm with their plus ends leading, and many of these are transported into the axon. In the schematic, the white portions of the microtubules represent the part assembled from the centrosome, while the black portions represent the part assembled after release from the centrosome. Plus ends of microtubules are directed away from the centrosome and toward the distal tip of the axon. The space between the slanted lines through the axon represents hundreds of micrometers of axon growth. During transit, the microtubules elongate specifically from their plus ends.

Still continues.......

Negative regulators of Neuronal polarity

2011-10-30T13:36:00.000+05:30

Negative regulators of neuronal polarity means the molecules will inhibit other downstream or upstream molecules which are involved in controlling the Cytoskeleton formation, stability, endocytosis etc.

PTEN (Phosphatase and tensin homolog)

It is a lipid phosphatase protein which counteracts the enzyme PI3 K (PI3 kinase) by converting PIP3 formed by PI3K into PIP2. Overexpression of this molecule will lead to prevention of PAR 3 localization to neurites and inhibits axon formation. If the PTEN expression is downregulated using SiRNA, then all the neurites becomes axons and there will reduction in dendrite formation. From this we can conclude that PTEN is important for the growth specific single axon. PAR 3 (Drosophila epithelia and neuroblasts) actually directly interacts with PTEN and this property leads to negative feedback loop. Where the PI3Kinase will recruit PAR 3 which in turn brings the PTEN molecule, thus leading to maintenance of PIP3 concentration in axon or PIP2 formed will bind to actin associated proteins such as WASP and regulate the actin formation.

PTEN is associated with PI3K and GSK3β; GSK3β inhibits PTEN by Phosphorylating at its C terminus. Until GSK3β is active, the PTEN activity is downregulated and therefore upregulation of PTEN happens by GSK3β inhibition which might provide a negative feedback regulation by again counteracting at PI3K activity.

Rho A (Rho GTPase Family)

Rho A is a Rho GTPase family protein which has the negative regulation role in the axon stability. Rho A negatively regulates the neurite outgrowth and modulating its signaling pathway will lead to axon regeneration in the central nervous system during injury. ROCK which is a Rho kinase effector accompanies Rho A in regulating axon growth and actin dynamics during neuritogenesis of cultured cerebellar granule neurons. Rho A-ROCK pathway with profilin II regulates actin and microtubule stability. Still Rho A knock mice is not created inorder to analysis the function of Rho A in neuronal polarity.

GSK3β (Glycogen synthase kinase 3β)

GSK3β is negative regulator of CRMP -2 (collapsin regulated mediator protein-2) which is GSK3β substrate responsible for microtubule polymerization. GSK3β is regulated by Akt protein which is activated by the PIP3 produced by PI3 Kinase. The protein not involved in establishment of neuronal polarity, but also plays a vital role in maintenance of neuronal polarity. CRMP-2 will be phosphorylated at thr-514 by GSK3β thereby making it unable to bind with tubulin dimer and NUMB. Overexpression of active GSK3β will lead to prevention of axon formation, whereas in inactive form it will lead to multiple axon formation. It also inactivates the MAP (microtubule associated proteins) protein by Phosphorylating MAP1B and APC (adenomatous polyposis coil).

Neuronal Polarity Introduction

2011-10-13T16:05:00.000+05:30

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.

Cytoskeleton Role in Neuronal polarity

2011-10-13T16:04:00.000+05:30

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:

The Microtubules rapidly convert the molecular signals into structural changes modulating cell shape.
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.

Actin and Microtubules regulators

2011-10-12T16:05:00.001+05:30

Actin regulating proteins

Basically the actin dynamics are regulated by actin nucleating, severing, branching and bundling proteins.

Actin nucleating proteins are Arp 2/3 complex (Lamellipodia) and formins (Filopodia). The Arp 2/3 complex is also very important in the formation of Filopodia as well as dispensable for actin organization in neuronal growth cones.

Growth cone dynamics regulators (directly modulate actin dynamics):

WAVE (wiskott Aldrich syndrome protein [WASP] family verprolin homologous protein)
Ena/VASP (Vasodilator stimulated phosphoprotein)
Profilin
ADF (actin depolymerization factor)/ cofilin

WAVE protein is localized in the Lamellipodia and is part of the WAVE complex of the proteins that act together to regulate actin polymerization in Lamellipodia. Basically the WAVE pathway regulates the actin polymerization through the Arp 2/3 or Profilin and promotes axon growth.

A component of WAVE complex called Nap 1 (Nck associated protein 1) which is important in cortical neuronal differentiation as well as proper extension of axon.

The Ena/VASP proteins are localized at the tips of the Lamellipodia and Filopodia, which will accelerate the actin polymerization by their anticapping activity and bundle actin filaments. Absence of these proteins will lead to aberrant actin bundling and failure of Filopodia formation. Therefore formation of neurites in the absences of these proteins is not possible, hence it’s considered important in the neuron formation.

The role of Ena/ VASP signaling in axon formation appears to be conserved throughout the species. Additionally, Ena/VASP proteins recruit the actin regulator profilin, but the physiological relevance of this interaction is still not clear.

Profilin localizes to the leading edges of the growth cones and enhances the formatting of ATP bound G actin monomers that are incorporated into actin barbed ends. Ablation of isoform Profilin IIa protein leads to destabilization of actin cytoskeleton as well as increased in the number and length of the neurites.

But it was investigated in the Profilin Knockout mice, that polarization has taken place normally. It shows that Profilin I is compensating the function of Profilin II, But Profilin I knockout mice are embryonical lethal.

Cofilin is an actin severing proteins which are also implicated in the neuronal polarity. ADF and cofilin І which are expressed in brain is found to be abundant in the neuronal growth cones. They bind mostly to the ADP-actin rather than ATP actin which results in their association with the pointed end of actin filaments and promoting actin depolymerization. Both the proteins are regulated by Phosphorylation and phosphorylated form is mostly abundant in the cellular forms. The cofilin proteins are active during axonal growth cones compared to non growing future dendritic growth cones.

Microtubule regulating proteins

CLIP (Cytoplasmic linker proteins) and CLASPs (CLIP associated proteins) regulated the microtubule dynamics. They act as MT growth promoting and also MT stabilizing protein.

CRMP 2 (collapsin response mediator protein 2) binds to the free tubulin and promotes their capacity to bind to the microtubules. CRMP2 is inhibited by GSK 3β Phosphorylation and PTEN. WAVE complex helps the transportation CRMP2 on kinesin molecule.

Stathmin/Op18 is the MT destabilizing proteins, which can be inactivated inorder to attain MT stability and is necessary for the specification of the nascent axon.

Neuronal polarity regulation

2011-10-12T14:20:00.000+05:30

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.

PAR protein in axon specification and neuronal polarity

2011-10-11T21:59:00.000+05:30

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.