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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Centrosome role in neuronal polarity

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.......

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Negative regulators of Neuronal polarity

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).

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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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