Transcript Denervation and Regeneration of Synaptic Connections

Cytoskeleton of Neuron
and Glia: Denervation and
Regeneration of Synaptic
Connections
Neurochemistry 10-04-2007
Jin-Chung Chen
Functions of the cytoskeleton proteins
Neurons could not divide, their distinctive
morphologies are maintained throughout life.
1. Provide structural organization and establish
metabolic compartments.
2. Serve as tracks for intracellular transport, which
creates and maintains differentiated cellular
functions.
3. Comprises the core framework of cellular
morphologies.
Molecular Components of the Neuronal
Cytoskeleton
The cytoskeleton is one of sevreal biological
elements that define eukaryotic cells (others
include the nucleus and mitochondria)
1. Microtubules (MTs)
2. Neurofilaments (NFs)
3. Microfilaments (MFs)
Microtutules act as both dynamic structural
elements and tracks for organelle traffic
1. Core structure: a polymer
of 50kDa tubulin subunits.
2. Heterodimers of - and tubulin align end-to-end
to form protofilaments.
3. 13 (also 12 or 14) of
which join laterally to
form a hollow tube with
an outer diameter of 25
nm
5. Tubulin dimers bind 2
GTPs and exhibit
GTPase activity, linked
4. 40% sequences similarity with assembly and
disassembly.
between - and -subunit
Tubulin of  dimer contains both “plus” and
“minus” end.
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
Organization of MTs in neurons
1. Axonal and dendritic MTs are not
continuous back to the cell body nor are
associated with MTOC.
2. Axonal MTs can be more than 100 m
long.
3. Uniform polarity, with all plus-end distal
to the cell body.
4. Dendritic MTs are typically shorter and
often exhibit mixed polarity.
Post-translational modification of tubulin
1. -tubulin can be modified by either Cterminal 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
Brain MT-associated proteins (MAPs)
1. MAP1a: preferentially in dentrites; modified by
phosphorylation
2. MAP1b: appears early then declines, enriched in
axons; modified by phosphorylation
3. MAP2a: high molecular weight, dendritic in
mature neurons; modified by phosphorylation
4. MAP2b: high molecular weight, dendritic
expressed throughout lifetime
5. MAP2c: Low molecular weight, dendritic in
developing neurons
….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
Neurofilament (neuronal and glial intermediate
filaments): support neuronal and glial mophologies
1. Five different types
2. Multiple -helical
domains, coiled/coils
3. Structually form 8-10
nm rope-like filament
4. Type I/II: keratins;
mainly in epithelial
cells throughout the
body
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)
Actin microfilament and 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
MF-associated proteins in neurons
1. Tropomyosin: stabilizes MFs
2. Spectrin/fodrin: cross-link MFs in membrane
cytoskeleton; enriched in membrane
3. Ankyrin: links MF/spectrin to membrane
proteins
4. Fimbrin: MF bunding and cross-linking;
involved in growing neurites
5. Gelsolin: fragments MFs and nucleates assembly,
regulated by Ca2+ (growing neurites and glia)
…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
The cytoskeletal elements of a growth cone
are organized for motility
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
Growth cone: MF and MT dynamics
For neuron to make synapse:
1. First stage: neurite elongation and pathfinding.
2. Initiated by growth cone: interpret extracellular
cues to steer the growing neurite in the right
direction
3. Growth cone receive both attractive
(neurotrophins/matrix proteins) and repulsive cues
4. Three domains: filopodia (long, thin, spike-like
projection), lamellipodia (web-like veils of cytoplasm)
and body (adheres to the substratum)
II. Nerve Denervation
1. Most neurons are postmitotic; only a few exit as
neuroepithelial stem cells.
2. Neurons lost to injury or disease cannot be
replaced.
3. Nerve cells can regenerate severed axons and
dendrites
Degenerative changes after axotomy (NMJ)
There are 5 major events: Wallerian degeneration,
chromatolytic reaction, target atrophies/dies,
synaptic stripping and immune response
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
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
Effect of Axotomy on Presynaptic cells
(example of autonomic ganglia)
1. Decreased sensitivity to its nerve signals;
2. Presynaptic terminals retract from the
axotomized cells and release less transmitter
(transsynaptic retrograde effects)
3. Undamaged neurons innervating the original
neuron increase additional synapses (signal
spreading)
4. 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.
Effects of Denervation on the Postsynaptic cells
(model of neuromuscular junction)
1. After (3-5 days) severance of nerve supply,
individual muscle fibers might be atrophy or have
spontaneous, asynchronous contractions
(fibrillation)
2. Fibrillation caused by changes in the muscle
membrane (not by transmitter ACh)
3. Mammalian muscle fiber becomes supersensitive
to a variety of chemicals ( all the source of
incoming transmitters, stretch or pressure)
4. AP in denervated muscles change, become more
resistant to tetrodotoxin (due to reappearance of
TTX-resistant Na channels)
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)
Distribution and Turnover of ACh receptors in
denervated muscle (labeled with -bungarotoxin)
1. After denervation, density of ACh receptors
increase in the extrasynaptic region
2. Receptor increase is due to enhanced synthesis,
not to reduced degradation
3. ACh receptor genes (nuclei) are all along the
length of denervated muscle fibers
4. New receptors in denervated adult muscle
display an embryonic type of receptor ( rather
than  subunit)
5. Denervation increases the rate of receptor
degradation (close to the fast turnover of
embryonic muscle)
Synthesis and Distribution of ACh receptors
Mechanism of new receptor synthesis
1. Not due to the presence of trophic factor
2. Inactivates the nerve (local anesthesia) would simulate
the denervation and cause muscle supersensitivity
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
Denervation on postsynaptic neurons
1. 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)
2. Not all the postsynaptic neurons response the same: some
have no changes, and some lose the enzyme activity
Effect on non-neuronal cell: immune response
1. Axotomy of CNS neurons leads to the
activation of microglia and astrocytes
2. Both cell types participate in synaptic
stripping
3. Reactive astrocytes also can form a scar (glial
scar) near sites of injury
4. If immune response enhanced, the monocytes
and macrophages will be recruited
Denervation and Axon sprouting
(Regeneration issues)
1. Health muscle would not receive extra innervation,
but nerve fibers will reinnervate an injured muscle
2. During the development, growth cones contact
muscle randomly, but reinnervation (distal stump)
usually reaches the site of original end plate
3. For supersensitivity and innervation: both initial
innervation (embryonic stage) and reinnervation
occur when the muscles are supersensitive
(prerequisite).
4. The denervated muscles are not only amenable to
innervation, but induce nerve sprouting new
terminals.
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
Extracellular mateix
molecule promote
neurite outgrowth:
1. Matrix proteins: collagens,
fibronectin, proteoglycans
and most important the
heterotrimeric laminins
(14 trimers identified)
2. Signal receptors: integrins
(dimers: 16 and 8 form)
on the membrane of
growth cone binds with
matrix proteins and
activates associated
proteins intracellularly.
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
Nerve terminals sprout in response to partial denervation
1. Sprouting and
Reinnervation occur
if muscle activity is
prevented by blocking
AP or denervation
2. Similar mechanism
also occurs in
autonomic ganglia or
axonal projection in
brain
Synaptic Basal Lamina (NMJ)
1. A structure, specialized region of the extracellular
matrix, plays a key role in the regeneration of
synapses between nerve and muscle
2. The materials in basal lamina constitutes a dense
staining matrix of proteoglycans and glycoproteins
(includes collagens, laminin, fibronectin)
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
Components in the synaptic
basal lamina direct the
clustering of ACh
receptors on the muscle
surface:
1. Denervation and muscle
fiber elimination but
preservation of the basal
lamina
2. In the absence of the nerve,
ACh receptors cluster on
muscle fiber at the original
synaptic site
5. Both regenerating nerve terminals and regenerating
myofibers can form synapse by the help of basal
lamina
Significance of Agrin (growth cone
development and regeneration)
1. 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
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).
Axon regeneration in mammalian CNS
1. In general, re-growth of cut axons in the adult
mammalian CNS is quite restricted
2. Axons in the CNS can grow for distances of several
centimeters under suitable circumstances
3. 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
Factors contribute to superior regenerative
capacities of the PNS vs. CNS neuron
1. Peripheral nerve and Schwann cells are potent promotor
of neurite outgrowth (contains laminin, NCAM and
trophic molecules)
2. Central nerves contain inhibitory components (myelinassociated glycoprotein and neurite inhibitor of 35kDa are
potent inhibitors to axon outgrowth)
3. CNS neuron contains less of GAP-43
4. CNS has prominent immune environment: astrocyte
proliferation, activation of microglia, scar formation,
inflammation and invation by immune cells.
Stretagies to regenerate damaged neural function
A. Transplant Schwann cells promote growth
Reconnection of retina and superior colliculus through
B. peripheral nerve graft
A. 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
B. EM illustrate a regenerated retinal ganglion cell axon terminal
in the superior colliculus containing synaptic vesicles
C. Transplanting the embryonic tissues
Features of embryonic transplant
1. Transplant embryonic nerve cells into the adult brain
2. The transplanted neurons can differentiate, extend
axons and release transmitters
3. Clinic application: fetal midbrain neuron restore
dopamine activity in the basal ganglia (Parkinsonism)
4. Grafting embryonic tissue into lesioned adult cortex,
hippocampus and striatum would also make
appropriate synaptic circuitry
5. Best example: anatomical and functional integration of
transplanted cerebellar Purkinje cells in the adult PCD
(Purkinje-cell-degeneration)
Reconstruction of
cerebellar circuits by
transplantation of
embryonic Purkinje cells
into an adult pcd mouse