Glia and Myelination, Part 1 PDF

Glia and Myelination, part 1 Review for further reading: Franklin RJM, Ffrench-Constant C (2017) Regenerating CNS myelin – from mechanisms to experimental medicines. Nature Reviews Neurosci. 18(12):753-769. For Wednesday Edgar JM, McGowan E et al (2021) Rio-Hortega’s drawings revisited with fluores...

Glia and Myelination, part 1 Review for further reading: Franklin RJM, Ffrench-Constant C (2017) Regenerating CNS myelin – from mechanisms to experimental medicines. Nature Reviews Neurosci. 18(12):753-769. For Wednesday Edgar JM, McGowan E et al (2021) Rio-Hortega’s drawings revisited with fluorescent protein defines a cytoplasmFilled channel system of CNS myelin. Journal of Anatomy. 239(6):1241-1255. doi: 10.1111/joa.13577 Tim Kennedy, MNI BT108 [email protected] brain is mostly made 10-50 X more glia than neurons in CNS. of glia Glia: derived from the Greek word for glue. Glial cells surround and support neurons. they have more important functions as well 4 major classes of glial cells. ectodermal layer —> neruoectodoerm 1. Astrocytes (derived from neuroepithelium) 2. Microglia (related to macrophages, mesodermal) immune cells in CNS, come from mesoderm Myelinating glia 3. Oligodendrocytes (CNS, derived from neuroepithelium) 4. Schwann Cells (PNS, derived from neuroepithelium, neural crest) Different between CNS and PNS —> CNS is encased in bone, PNS is not. Retina is CNS. Astrocytes: star shaped cell bodies Long processes have end-feet that contact capillaries or neurons. Function to bring nutrients into the CNS and to prevent the entry of many compounds into the CNS. The end feet of astrocytes contribute to the blood-brain barrier. Also control extracellular potassium. Are able to take up large amounts of potassium released by neuronal activity. And contain large stores of glycogen and supply neurons with glucose and lactate. all potassium going out will depolarize other cells, so astrocytes suck out all potassium an intermediate filament, glial fibrilary acid protein (GFAP), is a marker for astrocytes ) Myelinating Glia Oligodendrocytes (CNS) and Schwann cells (PNS) dendrites are NOT mylenated. Most axons are mylenated. Provide an electrically insulating spiral membrane wrap around axons. This specialized membrane wrap is myelin. A defining characteristic of vertebrates. Allows axons to be a lot thinner, and allows electrical signal to jump from node to node. The node of Ranvier is a highly specialized region of the axonal membrane that is not myelinated. Voltage gated sodium channels are concentrated at the node, and it is here that the action potential is regenerated. Myelin was first described in 1854 by Dr. Rudolf Virchow (1821-1902) in Berlin. He is remembered as the founder of “social medicine”, the father of modern pathology, for promoting cell theory, and for encouraging his medical students to “think microscopically”. Schwann cells and PNS myelin. One Schwann cell wraps only one axon in the peripheral nervous system (PNS). Each segment of myelin along a peripheral nerve is about 1 mm long (one Schwann cell). distance between the nodes is about 1 mm long. Theodore Schwann, 1810 - 1882 Oligodendrocytes: CNS Myelinating Glia Unlike Schwann cells in the PNS, an oligodendrocyte will myelinate multiple axons simultaneously. On average, one oligodendrocyte myelinates ~15 axons. Internode length is variable, from 10s of microns to ~100 microns. Many perineural non-myelinating oligodendrocytes tile the CNS gray matter. Their function is essentially unknown. make new myelin if you need it - myelin plasticity! Make new myelinated segments Santiago Ramón y Cajal (1852 – 1934) self portrait,1885, age 33 a young professor in Valencia, in his lab which was also his kitchen Camillo Golgi, 1873, The Golgi stain "la reazione nera" the "black reaction" silver staining technique Btw 1880s – 1930s, major fundamental contributions 1. Resolved reticular theory vs the neuron doctrine- discovered the individual cells 2. Argued that neurons are polarized 3. Described synapses between neurons 4. Provided the foundation for cellular neuroanatomy 1906 (age 54): Nobel prize in Physiology and Medicine shared it with Golgi synapse, Oligodendroglia: resolution of an old controversy. Those two are who discovered oligodendrocytes Penfield - American doctor, learned from Rio-Hortega - developed metal staining for cells in neuro tissue What is the developmental origin of oligodendrocytes and Schwann cells? Where do Schwann cells come from? Neural crest: Born in neural epithelium and migrate out of the neural tube (CNS) to myelinate axons in peripheral nervous system (PNS). Where do Oligodendrocytes come from? Born as oligodendrocyte precursor cells (OPCs) in the ventricular zone at several places along the neural tube, and then migrate and proliferate to populate all myelinated regions of the CNS. Oligodendroglial development in the mammalian spinal cord 1. OPCs born in ventricular zone dorsal 2. OPC Migration to where axons would be 3. OPC Proliferation cells are overproduced and then die ventricular zone ventral 4. Differentiation to post-mitotic but pre-myelinating OLs. 5. Establishment of contact with axons, myelin formation and elimination of superfluous OLs by apoptosis. motile, mitotically active cell - looking for cells to myelinate. Red stuff is acin, which there is lots of. PDGF alpha receptor - one thing that drives their proliferation is PDGF, and here its a marker migrating oligodendrocyte precursor cell Jarjour and Kennedy, 2004 Hoechst F-actin PDGFR (OPC marker) Oligodendroglia development in the mammalian spinal cord dorsal Oligodendrocyte precursor Cells (OPC) are born in the ventral midline of the embryonic spinal cord, just dorsal to the floor plate. They then migrate dorsally away from the floor plate (FP). What directs OPC migration? NETRINS Noto: notochord MN: motoneurons What directs OPC migration? from Orentas and Miller, 1999 ventral Netrins: Secreted Chemotropic Guidance Cues for Cell and Axon Migration ECM - you get polarity by secreting ECM from one cell, secreting one protein. Netrins are a family of secreted proteins related to laminins. Netrins are most well known for their function as axon guidance cues during neural development. Netrins are ~75 Kda. Netrin-1 is made in the floor plate at the ventral midline of the developing neural tube. Secreted netrin-1 forms a ventral to dorsal gradient. Laminins are large, ~800 KDa heterotrimeric extracellular matrix proteins. Best known for forming basal lamina, hence the name laminins. NEtrins can attract neurons and repel other neurons. Immunoflorescence, spinalJN cord Kennedyembryonic et al., 2006 Netrin-1 red Neurofilament M, NFM, green DCC and UNC5 families of netrin receptors. DCC is required for attraction to netrin-1. UNC5 homologs (UNC5A-D) are required for repulsion to netrin-1. Some cells make both DCC and an UNC5 and have the capacity to respond to netrin-1 as an attractant or a repellent depending on the cellular context. OPCs express DCC and UNC5A & B but not netrin-1 there are for UBC5 homologues. Netrin-1 Signal Transduction Chemoattraction Chemorepulsion very little is known Chemoattraction: Netrins regulate cytoskeletal organization to direct migration. Netrins also influence cell-substrate and cell-cell adhesion. Don’t memorize the details on this slides. Netrin-1 repels oligodendroctye precursor cells away from the ventral midline, directing them toward axons in what will become the white matter. Oligodendroctye precursor cells do not express netrin-1 but do express DCC and UNC5A netrin receptors. netrin-1 OPCs are repelled away from netrin Jarjour AA et al 2003 Long range - have to travel a lot SHort range: made locally and used locally Multiple extracellular guidance cues direct OPCs migration Long Range Repellents: Netrin, Semaphorin Jarjour and Kennedy, 2004 Organization of Myelin and Specialized Axonal Domains internode, compact myelin COncentrated Na+ channels Sodium Channels (Nav) Caspr gNetrin-1 DCC knockout micepotential die at birth. saltatoryand conduction of action from node to node compacted myelin —> all of paranodal loops are interconnected in the cytoplasm - protein on axonal surface, it is part of the junction, and you will see surface of axon at that junction Potassium Channels (Kv) Compacted and Non-compacted Oligodendrocyte Membranes all red is myelin-basic protein (MBP) which marks myelin membrane call body Green is type I transmembrane protein associated with noncompact membrane The major axis of polarization of a myelinating glial cell is the cell-body verses the myelin membrane. The myelin sheath is sub-divided into compact and non-compact membrane regions. Image is a single mature oligodendrocyte in culture without a axon. Red is immunostaining for myelin basic protein (MBP). MBP marks compact myelin membrane (internode). Green is TMEM10, a type I transmembrane protein that is not associated with compact myelin. The image illustrates the molecular specialization of the compact myelin vs the non-compacted oligodendrocyte membranes. Model of Myelin Formation during Development Illustration of the generation of the spiral wrap of membrane, followed by compaction and formation of compact myelin. Similar process for Schwann cells (PNS) and oligodendrocytes (CNS). pair of plasma membranes Schwann cells g-ratio = axon diameter / total outer diameter always less than one - unless it will be 1 when you normal (optimal) ~ 0.6 – 0.8 it’s have no myelin. Normal ratio = 0-.6-0.8 The optimal g-ratio is maintained for axons of different diameters. Thicker axons have thicker myelin. Think axons have thin myelin. of Development of Myelin Myelin compaction in the developing PNS (1950s EM). Ax: Axon SM: Schwann cell outer membrane Sc Cyt: Schwann cell cytoplasm OM: outer mesaxon IM: inner mesaxon/inner tongue MI: compact myelin The composition of compact myelin is similar to plasma membrane: ~80% lipid, ~20% protein, with high concentrations of cholesterol and glycolipids like galactocerebroside. Sphingomyelin is a specialized sphingolipid composed of sphingosine (lipid) linked to phosphocholine (hydrocarbon and nitrogen) that is enriched in myelin. Hydrocarbon chains extending from the lipid bilayer strengthen myelin. paranode runs around the edges Model of Myelin Biogenesis in the CNS: (A–D) Model of a developing myelin sheath in wrapped, unwrapped, and cross-section views. The unwrapped representation shows the geometry and the development of the sheath and the localization of cytoplasmic channels, that connect the cell body and the growth zone at the inner tongue. The growth zone is colored in pink, and the compacted myelin is dark violet. The wrapped representation shows the position of the layers when wrapped around the axon. The crosssections show the state of compaction during myelin growth. oligodendrocytes will make myelin wraps without a living axon myelinated nano‐fiber assays Bullock & Rome (1990) Glass micro-fibers: a model system for study of early events in myelination. J. Neurosci. Res. 27, 383–393. 5um diameter glass fibers. J Chan lab: Lee et al (2012) A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nature Methods 9, 917-922. Bechler, Byrne, ffrench-Constant (2015) CNS myelin sheath lengths are an intrinsic property of oligodendrocytes. Current Biology 25, 2411–2416. (Lee et al 2012) (Bechler et al 2015) Live‐Imaging Myelination in 3D 8h, 2min intervals, 5 fields 1 DIV 1h, 2 min intervals. 3 fields 1 DIV • OLs transfected with fluorophores (shown here; farnesylated eGFP) or dyed for live imaging • Zeiss 880 Ayriscan fastscan confocal imaging. 2 DIV Daryan Chitzas oligodendrocyes DON’T need a specific signal from axon to make myelin myelinating nanofiber cell culture compac ted lipid bilayer Proteins associated with compact myelin MBP is membrane ASSOCIATED protein -it helps with bringing two phospholilips membrane faces together. Acts like a molecular bridge. genetic basis for sharkomary tooth disease in PNS Myelin basic protein (MBP) ~30% and proteolipid protein (PLP) ~50% of total protein in PNS and CNS compact myelin. PLP is MOST abundant protein in myelin. function of PLP is to use extracellular loops is to link the two membranes together =MBP two phospholipid membranes that are zipped together Molecular structure of the myelin sheath. PLP is responsible for linking and compacting the extracellular face of the plasma membrane. PLP is translated at the rER in the cell body, and transport vesicles traffic it to the plasma membrane. Also evidence for transcytosis of PLP, trafficking it first to the cell body plasma membrane. It is then retrieving by endocytosis, and stored in a late endosomal compartment. Contact with an axon to be myelinated then triggers recruitment to the plasma membrane. In contrast, mRNA encoding MBP is transported along microtubules to the cell perimeter as an mRNA transport granule in which translation is suppressed. Translation occurs locally, near the point of insertion of MBP protein into the plasma membrane. MBP protein leads to dissolution of the cytoskeleton and compaction of the cytoplasmic compartment. Localizing translation to the point of membrane insertion of the protein prevents inappropriate membrane compaction. Major risk factor for MS is vitamin D. Demyelinating Diseases Lost or abnormal myelin leads to a decrease in axon conduction velocity. Can produce major disruptions of neuronal function in the brain, spinal cord, and peripheral nervous system. Multiple sclerosis, for example. the cell that is destroyed in MS is oligodendrocytes. MBP, myelin basic protein, plays an important role in myelin compaction (CNS and PNS). Injection of MBP into a mouse causes experimental allergic encephalomyelitis (EAE), characterized by loss of CNS myelin. This is used as an experimental model of demyelinating diseases, such as multiple sclerosis. The shiverer mutant mouse has a deletion mutation of the MBP gene. This produces tremors, convulsions, and early death. Transgenic addition of a copy of the MBP gene, rescues the shiverer phenotype. Charcot-Marie-Tooth disease (CMT). The gene encoding Peripheral Myelin Protein 22 (PMP22) is duplicated in CMT, the most common inherited peripheral neuropathy. Disease caused by an increase in gene dosage. Characterized by cycles of demyelination and remyelination, weakness, and greatly decreased conduction velocity in the peripheral nerves. Jean-Martin Charcot (1825-1893) Salpêtière, Paris. Pierre Marie (1853-1940) Howard Henry Tooth (1856-1925) FISH (florescent in situ hybridization) Panel D and E are chromosomal in situ hybridization using a red probe for the PMP22 gene. Note that in E one chromosome derived from an individual with CMT has two copies of the PMP22 gene. in demylenating disorders, the eciting thing is you jsut have to teach the cells to replace the way they are meant to do. Transplantation of human oliogdendrocyte precursor cells leads to widespread myelination of the brain in a mouse model of leukodystrophy. Leukodystrophies: A group of genetic disorders that effects the development and maintenance of myelin. Images of immunodeficient shiverer mice transplanted with human oligodendrocyte precursor cells (OPCs) and labeled with antibodies to human nuclear antigen (a) and antibodies to the myelin sheathassociated protein myelin basic protein (b), which is absent in shiverer mice. Conclude: an adult mammalian brain can be repopulated by transplanted OPCs and myelinated. a b from Franklin and ffrench-Constant 2008 you can heal the shiver mouse (red) by injecting human OPCs to cellularly transplant them into the brain, where they will spread and myelinate the brain