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ROLES OF GLIA IN THE NERVOUS SYSTEM

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Jonah Chan
I. Glia A. Glia are GFAP+ B. Schwann cells and oligodendrocytes are myelin forming cells and are essentially identical 1) They evolved separatelythey are from different lineages a) Schwann cells come from the neural crest 2) The only difference is oligodendrocytes form multiple myelin wraps while Schwann cells only form one segment 3) They share many of the same myelin proteins and the nodes are very similar 4) When Schwann cells form myelin, they spread out along the axon and synchronously start forming wraps at equal distances and does this very rapidly C. Microglia are induced after injury 1) Recent work suggests that microglia are always dynamic, even before injury, and may be involved in pruning of synapses and shaping synapses D. Astrocytes promote synaptogenesis and act as neuromodulators 1) Astrocytes engulfs the synapse and are involved in their formation 2) Astrocytes also surround the BBB and are involved in the delivery of nutrients into the brain II. Schwann cell A. Schwann cells space themselves along the axons in the PNS and form myelin 1) Schwann cells will die if it is not adhering and during development, competition between cells will cause some to fall off the axon and die B. Its thought there are three classes of Schwann cells, but its not clear they are actually different 1) Perisynatpic SC reside at the neuromuscular junction 2) Non-myelinating 3) Myelinating 4) All three classes come from the neural crest C. Expresses ErbB3, a receptor, and P75 neurotrophin receptor 1) ErbB3 is a classic tyrosine kinase receptor activated by EGF 2) P75 is part of the TNF receptor superfamily with a Death domain. But it doesnt seem to induce death; its involved in myelin formation. a) There are no known signaling cascade associated with P75 but it does have a large scaffold and has a PDZ domain D. Perisynaptic Schwann cells 1) The Schwann cell extends and covers up the neuromuscular synapse 2) Chien-Ping Ko identified a monoclonal antibody 2A12 that labels Schwann cells. When the antibody is injected into mice, complement-mediated lysis destroys the Schwann cells a) After ablation, nothing happened for a week . There was a little bit of retraction of the nerve terminal and a slight weakening of the synapse b) In an experiment where nerve regrows after axotomy, the Schwann cells are responsible for the clustering of AchR in the NMJ by expressing Agrin.

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Printed May 6, 2013 E. Schwann cells 1) Neuregulin1 is the ligand for ErbB3; PDNF is the ligand for P75. These are responsible for the maturation of Schwann cells to transition to myelin-forming cell F. Myelin 1) In order to form myelin, the intracellular cytoplasm has to be squeezed out and the outer leaflets from two adjacent membranes has to fuse 2) MBP, myelin basic protein, binds to the phospholipids and allows the membranes to approach each other. It is on the extracellular leaflet. In EM, the major dense line (the darker line) corresponds to the extracellular leaflet 3) During myelin formation, the cell body doesnt move. The new myelin forms against the axon, so it wraps from the inside a) However, how is it possible for the outside myelin to get larger and lager in diameter b) In addition, the axon swells when it is wrapped. So the problem is that the myelin layers also have to expand c) Not sure how fast this occurs. In 48 hours, there is something around 25 wraps formed. 4) Three questions arise: a) What are the cues that initiate development b) There must be polarity involved. When the myelin grows, it wraps in the same direction c) There must be a molecular motor. But because of the compactness of the membrane, there is no cytoplasm, no actin, no tubulin G. Axonal control of myelination 1) Since some axon are myelinated and some are not, its dogma that axons controlled myelination a) In 1989, Jim voyvodic demonstrated that when normally small diameter neurons were transplanted to a larger target and hypertrophied, it became myelinated b) However, this does not definitely show that axon diameter is responsible for myelination. There can be factors correlated with axon diameter 2) Its thought that NRG1 expressed on the axon membrane. The bigger the axon, the more NRG1 is expressed. This signals to ErbB3 on Schwann cells to begin myelination a) The more NRG1 is expressed, the more myelin is formed b) However, the domain that is responsible for ErbB3 signaling is cleaved of into a soluble form by other NRG isoforms. Only the membrane-anchored version is responsible for myelin formation 3) Cell polarity usually means that one side of the cell has different proteins from the other side of the cell a) This is asymmetry is done from Par proteins, Partitioning Defective family b) Par3 has a aPKC binding site, a GEF binding site that binds GEF which binds Rac1, and a PDZ domain that binds Par6. Par6 has a domain that binds Rac1 c) The Rac family of proteins belong with Rho

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Printed May 6, 2013 4) When axons form, the cell starts out with many processes. And these processes compete until one gets longer and wins 5) In culture, Schwann cells seeded onto neuron cultures form myelin after induction with ascorbic acid (vitamin C allows the formation of triple-helical collagen to form lamina) a) Par3 is localized to site of axo-glial junctions. b) When Par3 is knocked-down with shRNA, myelin does not form even though its correctly spaced out along the axon c) Par3 co-IPs with p75 right during the initiation of myelin formation d) P75 has a PDZ binding motif that may bind to Par3s PDZ motifs. Overexpression of Par3s PDZ domains blocks myelin formation e) So Chans idea is that Par3 helps enrich p75 to the inner membrane, allowing it to specifically interact with the NRG1 expressed on the axon and not the secreted ones III. Two questions: A. Why do we have two cell types that form myelin that evolved separately? B. If you had to design a structure like myelin, how would you do it? How is myelin formed?

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Oligodendroglia (Angela Hahn in Jonah Chans Lab)

I. Oligodendroglia are either the ligodendrocyte precursor cells or the differentiated premyelinating oligodendrocyte A. These are the myelinating cells of the CNS and form up to 100 different myelin segments per cell B. Axons are essential for the formation of myelin; just a stain for MBP does not demonstrate the presence of myelin C. OPCs are derived from oIPC (intermediate precursor cell) that are derived from radial glial cells 1) Oligodendrocytes do not start differentiating until the neonatal stage D. OPCs come from the same pool of cells as motor neurons 1) Motor neurons ar emade E9 to E10.5; OPCs are made E12.5 2) The motor neuron pool (pMN) are specified from the Shh gradient in the neural tube 3) Olig2 specifies OPCs instead of motor neurons when it is phosphorylated a) The switch takes 2 days to turn on and is irreversible E. OPCs are the only proliferating cells in this lineage 1) The mitogen is PDGF 2) Differentiated cells cannot proliferate anymore 3) Raffs group has data suggests that the oligodendrocytes have an internal timer that counts cell divisions which determines when the OPCs differentiate (Temple, 1986) 4) Later on, the same group (Gao, 1997) found that cells grown at 33C had fewer cells overall but more differentiated cells, suggesting that its not counting the number of cell divisions 5) Richardsons group showed that PDGF overexpression in mice makes more OPCs (because PDGF is responsible for proliferation), but the rate of differentiation is the same. However, the differentiated cells later die off to make a normal amount of myelin-makin cells. So there seems to be an extrinsic cue 6) Tang et al. (1999) showed that p27 (which arrests cell cycle) fails to promote differentiation. So stopping division is not sufficient either F. During differentiation, there is a major change in the plasma membrane as its morphology changes. 1) MBP is the only protein that is absolutely necessary for compacted myelin. 2) Its unknown what causes differentiation. Knocking out a variety of proteins doesnt stop differentiating and overexpressing proteins do not induce differentiation 3) However, a number of proteins, such as Jagged, Wnt, and CTGF prevents differentiation G. Myelination 1) The number of myelin wraps may be controlled by the neuregulin signalin pathway 2) Around the node of Ranvier, the ends of the myelin segment are perinodal loops. There is still cytoplasm in these loops

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Printed May 6, 2013 3) The myelin is replaced on the inner wrap. So new myelin is formed from the inside 4) Since all myelin forms on axons, axons must be involved in the formation of myelin. However, when the axons are plated and then fixed, myelin still forms 5) In vivo, the length and numbers of internodes are variable H. Between 2-9% of cells in the adult brain are undifferentiated OPCs. So, the adult brain could potentially remyelinate, if we can only induce myelination II. OPCs receive glutamatergic synapses A. There is a fast, large EPSC in oligodendrocytes. This is not just from spillover from the synapse since the concentration of glutamate required to elicit this EPSC is very high B. You can also observe mini EPSCs from OPCs as well and the current is blocked by traditional iGluR blockers like cadmium C. The current is blocked by AMPAR blockers D. Under EM, traditional synaptic structures are seen in NG2-neuron junctions E. In addition, OPCs receive synaptic input from axons in the white matter as well F. OPCs express TTX blockable sodium channels, but at a lower density G. While OPCs divide, the synapses are still functional. However, once the OPCs differentiate, the synapses stop functioning. 1) So perhaps the purpose of neuronal signaling is responsible for OPC differentiating? H. OPCs receive synapses from GABAergic cells as well, and because of the chloride current, the GABA signal is depolarizing

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Microglia (Dimitrios Davalos)

I. CNS Protection A. The CNS is immunoprivileged in that really nothing goes in and out. But if something does go wrong, microglia are there for protection B. Microglia are CNS macrophages 1) Glial cells are involved in tissue surveillance, homeostasis, trophic suppot, neurotransmitter clearance, synaptic pruning, synapse elimination 2) Mononuclear phagocytes release proinflammatory factors sucha s ascytokines and chemokines and are activated after injury to perform phagocytosis C. Microglia are still very controversial 1) Whats their origin? 2) Are there subtypes? 3) Are there really microglia? Or are the peripheral macrophages that come in? 4) Is observed behavior in vitro the same as in vivo? 5) What are their functions and activations tates? 6) Do they help in disease and injury? D. Adult microglia derive from primitive macrophages 1) Ransohoff (2010) showed that mesodermal monocytes become microglia and infiltrate CNS early during development 2) There are resident macrophages in the perivascular space, the choroid plexus, and in the meninges. But its not clear if there are resident microglia elsewhere E. But there are many types of microglia 1) There are amoeboid ones, parenchymal (resting) ones, reactive ones, and perivascular ones 2) When microglia are activated, they withdraw their processes and start migrating and behaving like microphages F. Its hard to study microglia in vitro because this obviously requires injury. So hes currently using in vivo maging in animals expressing fluorescent proteins using two-photon microscopy and think skull window techniques G. Fractalkine is expressed in activated endothelial cells and the receptor CX3CR1 is expressed in monocytes and microglia. The gene was replaced with the GFP ORF 1) Microglia cover 5-20% of all brain volume 2) Microglia processes essentially cover everywhere and the processes dont overlap or touch 3) The microglia processes extend and retract very rapidly to survey the brain for trauma 4) Upon injury, microglia extend processes towards localized injury and retract processes from opposite directions a) Signaling through ATP and P2X and P2Y. Blocking the P2Y receptor completely blocks microglial response b) Requires the release of ATP from astrocytes. Astrocytes have ATP stimulated ATP release H. The retraction of the microglial processes is signaled through Adenosine A2A receptors I. Microglial activation goes on the order of seconds to weeks [File] 6

Printed May 6, 2013 1) So there must be differen signaling pathways involved 2) The acute activation is probably aused by ATP and NO 3) The chronic activation include the traditional inflammation molecules such as TGFR, TNFR, interleukins, etc. J. In addition, microglia is proposed to be involved insynaptic pruning and synaptic stripping. But theres no direct demonstration of this yet 1) In Ben Barress lab, they stained synapses for PSD95 and SV2 for functional synapses. They find that non-functional synapses are coated with complement and the complement pathway signals to microglia to prune 2) Majewska showed using EM microscopy that theres more extracellular space around microglia and fewer s ynapses K. Microglia respond to various diseases, including Alzheimers, MS, Huntingtons prion, ALS, cancer, HIV, stroke, etc. 1) Mayer-Luhnmenn show that microglia move toward plaques in Alzheimers 2) But microglia seems to create more plaques when they go

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Neural-Glial Interactions in Regeneration (Jonah Chan)

I. Regeneration A. The major problem is that neurons rarely turnover and proliferate B. There seems to be positive factors in the PNS that support regeneration (or inhibitory factors in the CNS). If you transect the same neuron in the PNS, it will regenerate; but if the transaction is in the CNS, it will not 1) The extracellular matrix in the PNS is a contributing factor that allows regeneration C. Neurotrophin are factors that promote nerve growth and survival 1) NGF was discovered in chicks. Transplantation of a cancerous cell line into chicks caused hypertrophy of neurons near the transplant. The diffusible factor that caused the hypertrophy was NGF. 2) Shooter cloned and purified NGF. The 7S NGF is a protein made of 2, 2, and 2NGF subunits with 2 Zn2+ ions. The active subunit is the subunit 3) The neurotrophin hypothesis said that the target cell secreted NGF and the neuron will take up the NGF and cells that dont take up enough will die, creating competition 4) NGF will block axon degeneration after axonotomy 5) NGFs receptor is TRKA, tropomyosin receptor kinase, which is a tyrosine receptor kinase 6) Since NGF is a dimer, when it binds the receptor it dimerizes the receptor 7) Neurotrophins are those molecules that are homologous to NGF (BDNF, NT3, NT4/5) 8) Neurotrophins, besides bindin to the TRK family, also binds to p75 9) NGF signaling occurs when NGF binds to Trk receptors and the receptor/ligand complex is endocytosed and the activated Trk is able to interact with cytoplasmic second signaling molecules after endocytosis. 10)The four neurotrophin family members bind speicifc Trk receptors a) NGF binds TrkA b) BDNF binds TrkB c) NT3 binds TrkC d) NT4/5 binds TrkB e) And they all bind p75 f) The intracellular domains of TrkA/B/C are very similar; the ligand binding domain is the variable portion. g) Each of these receptors are alternatively spliced. There are truncated versions without the intracellular domain that will sequester the ligand without causing signaling. 11)Different types of neurons use different neurotrophins for survival D. Axon growth cone movement is based on the remodeling of the cytoskeleton. Inhibition of growth occurs throught he collapse of the growth cone 1) The cytoskeleton remodeling occurs through kinesin 2) Gomis-Ruth (2008) show that you can get multiple axons on a neuron if you stablilize the microtubules 3) The Rho GTPases regulate actin polymerization [File] 8

Printed May 6, 2013 E. Inhibitors of regeneration include 1) Ligands: Nogo-A, NOGO-66, MAG, OMgp 2) Receptors: Nogo Receptor, p75NTR 3) MAG and OMgp are myelin associated protein, which is where glia come in F. NOGO-66 binds MAG and OMgp 1) However, Nogo receptor has no intracellular domain, so downstream signaling is unknown 2) P75 interacts with Nogo Receptor and possibly p75 is what is signaling through RhoA to collapse the growth cone G. MAG is also expressed in Schwann cells, but Schwann cells dont block axon growth 1) MAG inhibition can be overcome by neurotrophins H. Glial scar is also a big problem in regeneration 1) A lot of this is because eof extracellular molecules, including CSPG (condroitan sulfate proteoglycans), NG2, MAG, and NOGO 2) Astrocytes are responsible for the scar.

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Astrocytes in Synapse Formation and Function (Nicola Allen)

I. Astrocytes A. Each astrocyte can contact up to 50,000 synapses B. Allen and Barres (2009), Eroglu and Barres(2010) C. Astrocytes touch blood vessels and synapses, allowin them to transport nutrients to neurons D. Astrocytes are known to be responsible for ion homeostasis, neurotransmitter uptake and recycling, energy and metabolite supply to neurons, and releasing neuronal survival factors E. Astrocytes express neurotransmitter receptors and respond to neurotransmitters with increases in intracellular calcium and the release of neuroactive substances 1) Proposed functions include synaptic plasticity, synaptic scaling, controlling blood flow, axon pruning and synapse elimination, and synapse formation F. Retinal ganglion cells can be grown in the presence of serine or on an astrocyte feeder layer 1) The feeder layer is separated from the neurons by a membrane 2) In the presence of astrocytes, the neurons form more functional synapses 3) Thrombospondin is released by astrocytes and induces the formation of synapses a) Christopherson et al (2005) b) These synapses release glutamate, but do not contain AMPAR and so are silent 4) Eroglu et al (2009) found that the EGF-like domain of Thrombospondin was responsible for synapse formation a) The 21 subunit of the VGCC is a receptor for the EGF-like domain b) Blockin 21 blocks synapse formation and overexpression of 21 increases the number of VGlut2 synapses G. Astrocytes release lipoparticles and Mauch et al (2001) showed that astrocytederived cholesterol enhances quantal content and presynaptic efficacy 1) However, when neurons are plated at high density the enhancement goes away. So perhaps its just a nourishmen thing and theres no real signal there H. Astrocytes increase the surface level of AMPA receptors but not the total AMPA receptors 1) In addition, the surface receptors are clustered in synaptic sites 2) Identified Glypican 4 as sufficient to form functional synapses in the presence of thrombospondin. It is a gpi-anchored heparin sulphate proteoglycan protein 3) Has been shown to be a carrier for different morphogens 4) Glypican 4 increases surface expression of AMPAR and these cells have increased synaptic activity 5) Glypican 6 is also sufficien to induce GluR1 containing synapses 6) This clustering of the AMPARs seem to precede the formation of the structural synapse 7) Glypicans 4 and 6 are also necessary for synapse formation as knockdown reduces the number of synapses formed. 8) Loss of Glypican 4 and 6 are weaker synapses and smaller mEPSCs [File] 10

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Blood Brain Barrier (Richard Daneman)

I. Vascularature A. There is a very dense network of blood vesssels in th brain and every neuron comes in contact to make sure it gets essential nutrients and oxygen B. Blood vessels also form a nice niche for stem cells so stem cells form right next to blood vessels, which secrete survival molecules C. Througout the body, the major nerves run parallel to major blood vessels. D. Endothelial cells in the CNS 1) The endothelial cells form tight junctions that block the flow of blood to he brain 2) Theres low rates of transcytosis, moving molecules across the cells that make up the vessel wall 3) Express extrusion pumps Pgp that pumps molecules that do manage to cross back into the blood 4) Express selective transporters for specific nutrients into the brain E. Transplantation of gut endothelia into the brain forms tight barrier as well. So the brain is causing the formation of the BBB 1) AStrocytes have been proposed to be the signal to form the BBB F. Endothelial cells begin invading the cortex around E12. Pericytes cover the endothelial cells as soon as they start invading the brain 1) Astrocytes are only formed afterwards. So the timing of AStrocyte signaling is a bit off 2) Occludin is where the tight junctions form between endothelial cells. These are seen even before endothelial cells form a tube. So astrocytes cannot be responsible for all the energy source of the brain G. Pericyte deficient mice have a leaky BBB 1) However, some of the tracer dye remains in the vessels, so there are still BBB properties 2) The fewer the pericytes, the more permeable the vessels become 3) The morphology of endothelial cells in pericyte deficient mice is strange. There are excess folds of endothelial cells into the lumen. The tight junctions also do not align correctly to be parallel to the lumen 4) In pericyte deficient mice, theres also an increased amount of vesicle trafficking 5) Pericytes are not important for the induction of any BBB genes. Rahter pericytes block the expression of genes that make blood vessels leaky 6) These defects are rescued by coculture with pericytes, even without direct contact. The factors are secreted H. Since even without pericytes, the endothelial cells still express BBB genes, something else must be inducing these genes I. Possibly, neural stem cells induce BBB gene expression and pericytes modulate the BBB 1) They FACS purified endothelial cells from different tissues and looked at what sdifferent from them 2) Wnt signaling is only turned on brain endothelial cells 3) Beta Catenin is necessary for CNS vessel formation

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Printed May 6, 2013 4) Most of vascularture durin development form in Wnt expressin areas. So during neural tube formation, the endothelial cells will invade the area near the ventricle, where the Wnt signaling occurs 5) The same attractive signals that bring the Endothelial cells into the nervous system also induces expression of the BBB transporters

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Remyelination ()
I. Developmental myelination A. Shh from the floor plate and BMPs fromt eh roof plate determine developmental cell types B. During demyleination, the sodium channels are redistributed C. PLP is a structure component of myelin and in the absence of PLP, axons degenerate 1) Cnp1 is also involved in myelination but in this case the axon degenerates even though the myelin is intact. This suggests that its not the myelin sheath that provides support to axons but that the oligodendrocyte is maintaining the axon 2) Myelin is also required for fast axonal transport. PLP deficient mice have issues with axonal transport 3) After demyelination, if the axon is not remyelinated, the axon will die D. In multiple sclerosis, oligodendrocytes are attacked by autoimmunity and the loss of myelin leads to axon degeneration 1) In patients, there is a cycle of degeneration and repair so that some myelin is replaced after acute demyelinating episodes 2) After insult, the oligodendrocytes die and myelin degenerates 3) This activates an immune response which signals to oligodendrocytes to proliferate, which remyelinate the axons E. Failure of remyelination often is resulted from inadequate differentiation of oligodendrocytes 1) The oligodendrocytes proliferate, but are blocked at a pre-myelinating phase F. In cerebral palsy, there may be excitotoxic insult that leads to oligodendrocyte death and a fixed lesion 1) However, evidence is emerging that oligodendrocytes are present in the lesion but they are not maturing correctly G. Remyelination is not just repeating developmental myelination. In remyelination, the axon has seen myelin before, there is debris and cell death, the axon is not as dynamic as during development, and there may be inflammation H. Axin2 is a product of the Wnt pathway and is an inhibitor of the Wnt pathway 1) In MS plaques, the Wnt pathway is activated 2) Axin2 is expressed in immature stages of oligodendrocyte differentiation and is not expressed with PLP 3) In Axin2 knockout mice, there is delayed myelination and remyelination 4) XAV939 stabilizes Axin2 by inhibiting Tankyrase and accelerates remyelination I. Thus, remyelination failure is due to inadequate differneation of oligodendrocytes and the Wnt pathway is responsible for inhibition of oligodendrocyte differentiation J.

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