Cytoskeleton of Neuron and Glia: Denervation and Regeneration of Synaptic Connections

1. Cytoskeleton of Neuron and Glia: Denervation and Regeneration of Synaptic Connections Neurochemistry 10-04-2007 Jin-Chung Chen

2. Functions of the cytoskeleton proteins • Neurons could not divide, their distinctive morphologies are maintained throughout life. • Provide structural organization and establish metabolic compartments. • Serve as tracks for intracellular transport, which creates and maintains differentiated cellular functions. • Comprises the core framework of cellular morphologies.

3. Molecular Components of the Neuronal Cytoskeleton • The cytoskeleton is one of sevreal biological elements that define eukaryotic cells (others include the nucleus and mitochondria) • Microtubules (MTs) • Neurofilaments (NFs) • Microfilaments (MFs)

4. Microtutules act as both dynamic structural elements and tracks for organelle traffic • Core structure: a polymer of 50kDa tubulin subunits. • Heterodimers of - and -tubulin align end-to-end to form protofilaments. • 13 (also 12 or 14) of which join laterally to form a hollow tube with an outer diameter of 25 nm • 40% sequences similarity between - and -subunit 5. Tubulin dimers bind 2 GTPs and exhibit GTPase activity, linked with assembly and disassembly.

5. Tubulin of  dimer contains both “plus” and “minus” end.

6. Microtubule-organizing center (MTOC) 1. Plus end of -tubulin is the preferred end for addition of tubulin dimers. 2. The minus end grows more slowly at physiological concentrations of tubulin. 3. In glia and most other non-neuronal cells, the minus-ends of MTs are usually bound at the site of mucleation (slow; followed by a rapid growth phase) which is associated with the centrosome or pericentriolar complex of the cell, a site often called the MTOC. 4. Anchoring of MT minus-end helps establish the polarity of MTs, requires presence of -tubulin

7. Organization of MTs in neurons • Axonal and dendritic MTs are not continuous back to the cell body nor are associated with MTOC. • Axonal MTs can be more than 100 m long. • Uniform polarity, with all plus-end distal to the cell body. • Dendritic MTs are typically shorter and often exhibit mixed polarity.

8. Post-translational modification of tubulin 1. -tubulin can be modified by either C-terminal tyrosination (via tyrosine ligase) or via lysine-ε-acetylation (via acetyltransferase) 2. -tubulin is modified by polyglutamylation (via -carboxylase) protein modification will facilitate the polymerization and elongation of MTs

9. Brain MT-associated proteins (MAPs) • MAP1a: preferentially in dentrites; modified by phosphorylation • MAP1b: appears early then declines, enriched in axons; modified by phosphorylation • MAP2a: high molecular weight, dendritic in mature neurons; modified by phosphorylation • MAP2b: high molecular weight, dendritic expressed throughout lifetime • MAP2c: Low molecular weight, dendritic in developing neurons

10. ….continuous 6.Tau (high MW): peripheral axons with distinctive phosphorylation pattern (low MW): enriched in CNS axons, regulated by phosphorylatoin 7. MAP4: primarily nonneuronal, multiple forms, phosphorylation at mitosis 8. Katanin: enriched at MTOC, an ATPase, serves MT (release MT into the axon) 9. Stathmin: destablizes MT, regulated by phosphorylation

11. Neurofilament (neuronal and glial intermediate filaments): support neuronal and glial mophologies • Five different types • Multiple -helical domains, coiled/coils • Structually form 8-10 nm rope-like filament • Type I/II: keratins; mainly in epithelial cells throughout the body

12. 5. Type III: Vimentin (neural and glial precursors) GFAP (astrocytes, some Schwann cells) peripherin (subset of neuron in peripheral) Desmin (smooth muscle cells in vasculature) 6. Type IV: NFH (high MW NF, 180-200 kDa); NFM (middle MW NF, 130-170 kDa); NFL (low MW NF, 60-70 kDa): (most neuron, abundant in large neurons) 7. Type V: nuclear lamins (nuclear envelope) 8. Type VI: nestin (neuroectodermal precursors)

13. Actin microfilamentand membrane cytoskeleton play critical roles in neuronal growth and secretion 1. 43 kDa monomers; arranged into fibrils of 4-6 nm diameter 2. A remarkable variety of proteins interact with actin MF: myosin, spectrin, tropomyosin…etc. 3. Particularly concentrated in presynaptic terminals, dendritic spines and growth cones 4. Main component of membrane cytoskeleton

14. MF-associated proteins in neurons • Tropomyosin: stabilizes MFs • Spectrin/fodrin: cross-link MFs in membrane cytoskeleton; enriched in membrane • Ankyrin: links MF/spectrin to membrane proteins • Fimbrin: MF bunding and cross-linking; involved in growing neurites • Gelsolin: fragments MFs and nucleates assembly, regulated by Ca2+ (growing neurites and glia)

15. …continuous 6. -thymosins: binds actin and regulates MF assembly (growing neurites) 7. Profilin: inhibits MF formation, regulated by selected signaling pathway (growing neurites, glia) 8. Arp2/3 complex: nucleation of actin MF assembly in cortex and initiation of MF branches 9. N-WASP: interacts with Arp2/3 complex to nucleate actin MF assembly; enriched in cortex

16. The cytoskeletal elements of a growth cone are organized for motility

17. There are three domains of the growth cone: filopodia, lamellipodia and central core Actin is rich in lamellipodia and filopodia Microtubules are concentrated in the central core

18. Growth cone: MF and MT dynamics • For neuron to make synapse: • First stage: neurite elongation and pathfinding. • Initiated by growth cone: interpret extracellular cues to steer the growing neurite in the right direction • Growth cone receive both attractive (neurotrophins/matrix proteins) and repulsive cues • Three domains: filopodia (long, thin, spike-like projection), lamellipodia (web-like veils of cytoplasm) and body (adheres to the substratum)

19. II. Nerve Denervation • Most neurons are postmitotic; only a few exit as neuroepithelial stem cells. • Neurons lost to injury or disease cannot be replaced. • Nerve cells can regenerate severed axons and dendrites

20. Degenerative changes after axotomy (NMJ) There are 5 major events: Wallerian degeneration, chromatolytic reaction, target atrophies/dies, synaptic stripping and immune response

21. Wallerian Degeneration (distal segment) and Chromatolysis (proximal segment) Wallerian degeneration: Transmission fail; physical degeneration on axon; myelin sheath fragment/enveloped by phagocytic cells. Chromatolysis: Cell body swell and nucleus move to eccentric position and fragmented ER; overall increase in protein and RNA synthesis and changes the pattern of gene expression

22. Presynaptic and postsynaptic events Postsynaptic neurons: Target cells (muscle or neuron) atrophy and sometimes dies; postsynaptic responses are subtle. Presynaptic input neurons: Synaptic terminals withdraw from the cell body or dendrites of chromatolytic neurons and are replaced by the processes of glial cells (synaptic stripping), depress synaptic function Trans-synaptic effects: neuronal degeneration can propagate through a circuit in both anterograde and retrograde directions

23. Effect of Axotomy on Presynaptic cells (example of autonomic ganglia) • Decreased sensitivity to its nerve signals; • Presynaptic terminals retract from the axotomized cells and release less transmitter (transsynaptic retrograde effects) • Undamaged neurons innervating the original neuron increase additional synapses (signal spreading) • A method could simulate the axotomy: disrupt the trophic substance retrograde transport (such as anti-NGF Ab injection). Schwann cell could supply the lost NGF and even express NGF receptor.

24. Effects of Denervation on the Postsynaptic cells (model of neuromuscular junction) • After (3-5 days) severance of nerve supply, individual muscle fibers might be atrophy or have spontaneous, asynchronous contractions (fibrillation) • Fibrillation caused by changes in the muscle membrane (not by transmitter ACh) • Mammalian muscle fiber becomes supersensitive to a variety of chemicals ( all the source of incoming transmitters, stretch or pressure) • AP in denervated muscles change, become more resistant to tetrodotoxin (due to reappearance of TTX-resistant Na channels)

25. 5. Supersensitivity to ACh is due to altered distribution of ACh receptors in denervated muscles 6. Muscle membrane are uniformly sensitive to ACh (intact muscle only end plate region sensitive to ACh)

26. Distribution and Turnover of ACh receptors in denervated muscle (labeled with -bungarotoxin) • After denervation, density of ACh receptors increase in the extrasynaptic region • Receptor increase is due to enhanced synthesis, not to reduced degradation • ACh receptor genes (nuclei) are all along the length of denervated muscle fibers • New receptors in denervated adult muscle display an embryonic type of receptor ( rather than  subunit) • Denervation increases the rate of receptor degradation (close to the fast turnover of embryonic muscle)

27. Synthesis and Distribution of ACh receptors

28. Mechanism of new receptor synthesis • Not due to the presence of trophic factor • Inactivates the nerve (local anesthesia) would simulate the denervation and cause muscle supersensitivity

29. 3. Repetitive direct stimulation of muscles over several days caused the sensitive area to become restricted (only synaptic region is sensitive to ACh) (muscle activity itself affects supersensitivity) Reversal of supersensitivity in a denervated muscle by direct stimulation of the muscle fiber

30. Denervation on postsynaptic neurons • Consequence of the postsynaptic neuronal change is similar to muscle: extrasynaptic ACh receptors appear in the postganglionic neurons after vagus nerve denevation (but no changes in membrane potential or excitability) • Not all the postsynaptic neurons response the same: some have no changes, and some lose the enzyme activity

31. Effect on non-neuronal cell: immune response • Axotomy of CNS neurons leads to the activation of microglia and astrocytes • Both cell types participate in synaptic stripping • Reactive astrocytes also can form a scar (glial scar) near sites of injury • If immune response enhanced, the monocytes and macrophages will be recruited

32. Denervation and Axon sprouting (Regeneration issues) • Health muscle would not receive extra innervation, but nerve fibers will reinnervate an injured muscle • During the development, growth cones contact muscle randomly, but reinnervation (distal stump) usually reaches the site of original end plate • For supersensitivity and innervation: both initial innervation (embryonic stage) and reinnervation occur when the muscles are supersensitive (prerequisite). • The denervated muscles are not only amenable to innervation, but induce nerve sprouting new terminals.

33. Motors drive the growth cone: 1. Actin assemble at filopodium; 2. Vesicle fusion add membrane to the filopodium 3. Actin polymerized to push filopodium forward 4. Microtubule from central core advance 5. Cytoplasm collapses to create new segment of axon

34. Extracellular mateix molecule promote neurite outgrowth: • Matrix proteins: collagens, fibronectin, proteoglycans and most important the heterotrimeric laminins (14 trimers identified) • Signal receptors: integrins (dimers: 16 and 8 form) on the membrane of growth cone binds with matrix proteins and activates associated proteins intracellularly.

35. Semaphorins (ephrins) and neuropilins (receptor) guide growth cone by providing inhibitory signals Binding of semaphorins (in the metrix) to neuropilins (growth cone) causes growth cones to collapase

36. Nerve terminals sprout in response to partial denervation 1. SproutingandReinnervationoccur if muscle activity is prevented by blocking AP or denervation 2. Similar mechanism also occurs in autonomic ganglia or axonal projection in brain

37. Synaptic Basal Lamina (NMJ) • A structure, specialized region of the extracellular matrix, plays a key role in the regeneration of synapses between nerve and muscle • The materials in basal lamina constitutes a dense staining matrix of proteoglycans and glycoproteins (includes collagens, laminin, fibronectin)

38. 3. After denervation, damaged region phagocytized and terminal degenerated, leave only the basal lamina sheaths remained intact 4. Week later, new myofibers had formed within the basal lamina sheaths and contacted with regenerated axon terminals (at original synapse) and muscle regain twitches

39. Components in the synaptic basal lamina direct the clustering of ACh receptors on the muscle surface: • Denervation and muscle fiber elimination but preservation of the basal lamina • In the absence of the nerve, ACh receptors cluster on muscle fiber at the original synaptic site

40. 5. Both regenerating nerve terminals and regenerating myofibers can form synapse by the help of basal lamina

41. Significance of Agrin (growth cone development and regeneration) • Extract from the basal lamina contains active component, agrin that help the formation of synapse and induce ACh receptor aggregation 2. Agrin is synthesized by motorneurons, transported down to axons, and released into lamina to induce postsynaptic differentiation during regeneration 3. Agrin might bind to its receptor in the postsynaptic surface, and trigger intracellular events: phosphorylation of  subunit of ACh receptor that leads to ACh receptor aggregation

43. Neuregulin stimulates expression of the genes encoding the ACh receptors Neuregulins are synthesized and secreted by motor axons. Binding of neuregulin to erbB kinase (erbB2, erbB3 and erbB4) in the postsynaptic membrane activates transcription of ACh receptor genes via a cascade of protein kinases (ras/raf/ETS).

44. Axon regeneration in mammalian CNS • In general, re-growth of cut axons in the adult mammalian CNS is quite restricted • Axons in the CNS can grow for distances of several centimeters under suitable circumstances • The most importance for axonal regeneration is the immediate environment encountered by growth cones, provided by Schwann cells in periphery and astrocytes and oligodendrocytes in the CNS

45. Factors contribute to superior regenerative capacities of the PNS vs. CNS neuron • Peripheral nerve and Schwann cells are potent promotor of neurite outgrowth (contains laminin, NCAM and trophic molecules) • Central nerves contain inhibitory components (myelin-associated glycoprotein and neurite inhibitor of 35kDa are potent inhibitors to axon outgrowth) • CNS neuron contains less of GAP-43 • CNS has prominent immune environment: astrocyte proliferation, activation of microglia, scar formation, inflammation and invation by immune cells.

46. Stretagies to regenerate damaged neural function A. Transplant Schwann cells promote growth

47. Reconnection of retina and superior colliculus through B.peripheral nerve graft • Optic nerves severed and one was replaced by a segment of nerve. Regeneration tested by anterograde tracers injection or recording responses of superior colliculus neurons to light flashed on the light • EM illustrate a regenerated retinal ganglion cell axon terminal in the superior colliculus containing synaptic vesicles

48. C. Transplanting the embryonic tissues

49. Features of embryonic transplant • Transplant embryonic nerve cells into the adult brain • The transplanted neurons can differentiate, extend axons and release transmitters • Clinic application: fetal midbrain neuron restore dopamine activity in the basal ganglia (Parkinsonism) • Grafting embryonic tissue into lesioned adult cortex, hippocampus and striatum would also make appropriate synaptic circuitry • Best example: anatomical and functional integration of transplanted cerebellar Purkinje cells in the adult PCD (Purkinje-cell-degeneration)

50. Reconstruction of cerebellar circuits by transplantation of embryonic Purkinje cells into an adult pcd mouse