Kennedy Glia and Myelination 2023 part 2.pdf

Glia and Myelination, part 2 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 2 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] Compact myelin looks like a crystal. tem: transmission electron microscopy tiny dots - intermediate filaments bigger dots - microtubules Cedric S Raine (1984) Morphology of Myelin and Myelination. Myelin, P. Morell (ed) Note outer tongue, inner tongue, MDL, IPL, scale bar = 0.1 micron. all of these change the rate of conduciton, and you change the timing of the neural circuit which changes how the circuit works Myelin Plasticity 1. Myelination of previously unmyelinated axons, or partially myelinated axons 2. Elongation of existing myelin 2. Retraction of existing myelin 2. Thinner or thicker existing myelin. 3. Alteration of node of Ranvier de Faria O Jr, Pivonkova H, Varga B, Timmler S, Evans KA, Káradóttir RT (2021) Periods of synchronized myelin changes shape brain function and plasticity. Nat Neurosci. 2021 Nov;24(11):1508-1521. Organization of Myelin and Specialized Axonal Domains: Node of Ranvier and Paranodal Axo-Glial Junctions internode, compact myelin paranodal loops Sodium Channels (Nav) Caspr gNetrin-1 and DCC knockout mice die at birth. these attachement sites are waht make myelin look ‘alive’ Potassium Channels (Kv) CM Smooth ER node of ranvieer CM Glial and Axonal molecular markers for nodes, paranodes, and juxtaparanodes N of Ran Na+ chan MBP CASPR is fantastic antibody to label Paranodal junction f Potassium Channels 3 part protein Neurofascin-155 Contactin CASPR-1 Sodium Channels CASPR MBP CNPase Kv1.2 THis protein acts as molecular fence that keeps potassium and sodium channels away from eachother to stop short circuits bc positive current floods in at node of ranvier and out at juxtaparanode whole bunch of cytoplasmic openings (like plumbing) - it is a continuous channel that spirals thru the compact myelin, supports the myelin Schwann Cells Note microvilli (specific to Schwann cells) at node of Ranvier. microvilli Paranodal loops Alignment of Protein Subdomains. 2nd panel: Na channels are red, restricted to node. Schmidt Lanterman incisures, green, are labeled with an antibody against MAG, myelin associated glycoprotein. 3rd panel: K channels are green. They are localized distal to the caspr collar (red). CASPR clamps down on axonal surface Schmidt Lanterman incisures Schwann cell wrapping an axon to form a paranodal junction Schwann cell is migrating along the axon in a spiral (red) sodium channels are getting pushed down as the myelin wraps Confocal image of early myelination during Schwann cell development. Panel A: F-actin (red), axon (blue). Cartoon C illustrate sodium channels as red. Schwann cell has made ~ 2 wraps around the axon. The Schwann cell is crawling along the axon in the direction of the arrow, towards what will become the node of Ranvier. F-actin and associated proteins likely contribute to force generation that drives process extension and wrapping. Na channels cluster at the advancing edge of the axon, thought to be swept there by a molecular sieve of transmembrane proteins composed of Caspr, NF-155, and contactin. Development of myelin (co-culture of SCs and DRGs) Segregation of Na channels A, B, C, D: Na channels are green. caspr is red neurofilament (axon) blue K channels are green in E. The front of the moving Schwann cell moves the Na channels towards each other, until the two hemi-nodes fuse forming a node of Ranvier. C: early node formation where the paranodes are still loose. Note the spiral of caspr immunoreactivity. The Schwann cell is like a plow that scopes up Na channels and concentrates them in the node (D), while K channels remain juxtaparanodal. Consolidation of the Caspr spiral myelinating co-cultures of SCs and DRG neurons antibodies against Caspr, transmembrane protein in the axon. Caspr/Contactin/NF-155 complex. Panel A shows a single spiral where one Schwann cell has contacted an axon. In b, the extending fronts of two Schwann cells approach each other. Panel C illustrates that the process can be asymetric, one hemi-node can mature faster then its partner. Panel D shows a mature node. Peeling away the compact myelin (e) reveals consolidated spiral of paranodal loops. Note: Na channels are bigger than K channels. Na channels bind ankyrin, K channels do not. leading edge is not compacted yet wrapping in two directions, and then you have mature oligodendrite 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. Specific cell-cell junctions anchor paranodal loops to the axon and to each other there are specialized axo-glial junctions, tight junctions (autotypic tight junctions - two membranes of ONE cell, making junction with itself) Jarjour et al. 2020 Specific cell-cell junctions anchor paranodal loops to the axon and to each other Axoglial junction NF155: Neurofascin 155 CNTN1: Contactin Genetic deletion (knockout) of caspr, NF155, or contactin results in disruption of paranodal junctions and disorganization of the specialized domains along axons Caspr, NF115, or contactin knockout • Detachment of glial loops, followed by leakage of caspr out of the paranode • Breakdown of the boundary between the paranode and juxtaparanode (K+ channels) and the node (Na+ channels) • Deficits in coordination and balance • Reduced conduction velocity Autotypic tight junctions and Gap junctions link paranodal loops to each other Tight junction Tight junctions and Gap junctions form autotypic junctions that link the paranodal loops to each other The TJ protein Claudin 11 is also called OSP, oligodendrocyte specific protein Claudin 11 maybe has ability to wind and unwind Other factors that influence paranode formation and maintenance? Mature oligodendrocytes express netrin‐1, DCC and UNC5B. Does netrin‐1 have a role at paranodes? Sees that netrin is right on top of CASPR - so netrin lives right at paranodal junction Netrin-1 is expressed by oligodendrocytes and enriched at CNS paranodes 4 μm 1 μm Jarjour et al 2008 DCC is ALSO at the paranode but instead of being on CASPR, it is around the CASPR. it would be on paranodal loops DCC is expressed by oligodendrocytes and distributed at CNS paranodes Jarjour et al 2008 Disruption of paranode maintenance ‐/‐ ‐/‐ in DCC or netrin‐1 organotypic slice cultures Jarjour et al 2008 Specific cell-cell junctions anchor paranodal loops to the axon and to each other Axoglial junction Jarjour et al. 2008 DCC (and netrin‐1, not shown) is required to maintain axoglial parnodal junctions in the mature CNS. Caspr distribution slides along the axon. When you KO netrin, you will get leaking of CASPR + + Abnormal distribution of Na and K channels in ‐/‐ ‐/‐ netrin‐1 or DCC myelinated axons Na and K will leak when the netrin and DCC knockouts exist Jarjour et al 2008 Flnaking Lock sites -short DNA seq. and put them around a gene, it is meant to mark a specific axon Selective DCC deletion from mature myelinating oligodendrocytes in vivo Cre is an enzyme - recognizes Lock sites - way to recombine DNA Selective deletion of floxed allele by cell type specific expression of tamoxifen regulated cre recombinase Tamoxifen induction at 4.5-6 week of age mice that are expressing Cre in tamoxifen is a drug that myelinating Glia and expressing allows the Cre to translocate DCCflox/flox so only in myelinating into the nucleus glia will they do the recombination Bull SJ, Bin JM et al 2014. JN PLPcreERT mice: Doerflinger et al., 2003 this allows us to look at living mouse, and grow normally, and just delete the gene later, so that the mouse doesnt die. Disorganization of Paranodal Junctions due to Loss of DCC Expression by Oligodendrocytes % of paranodes 100 wild type PLPcreT +DCCflox/flox 80 60 40 20 0 normal 1 fault 2 faults 3-4 faults Average number of faults 2.0 *** 1.5 1.0 n=47, 3 animals /group 0.5 0.0 wild type PLPcreT+DCCflox/flox Optic nerve – 6 months post induction Bull SJ*, Bin JM* et al 2014. J N Oligodendrocytes HAVE to make DCC in order to stay organized and proper function Summary Loss of DCC from myelinating oligodendrocytes • Loss of oligodendrocyte expression of DCC results in: • Detachment of glial loops, followed by leakage of caspr out of the paranode • Increased myelin outfoldings and reduced compact myelin proteins • Deficits in coordination and balance • Reduced conduction velocity • Progressively worse myelin disruption with aging Current Working Model paranodal loop DCC = receptor for netrin adhesive-cytoskeletal interaction? ? ? axonal membrane nf155 caspr contactin DCC netrin ? Specific cell-cell junctions anchor paranodal loops to the axon and to each other Tight junction Cell type specific deletion of UNC5B from oligodendrocytes UNC5B cKO among neural cells, only oligodend. make UNC5B De Faria et al, THIS MAKES IT A GREAT DRUG TARGET UNC5B deletions from OLs results in everted paranodal loops Reduced levels of tight junction protein claudin 11. Disruption of loop‐loop autotypic junctions. Is Unc5B requires to maintain paranodal loop‐loop tight junctions? TEM tomography GAPS IN COMPACT MYELIN - WHEN YOU DELETE UNC5B YOU DISTRUPT SOME OF THE CONNECTION BETWEEN THE PARANODAL LOOPS Is UNC5B a component of inter-loop junctions? UNC5B / CASPR 90o rotation 1 μm distribution of UNC5B - we don’t know yet SIM super resolution imaging Diane Nakamura If you take CS fluid - hypothesis is - you would see compact myelin protein in the CSF of the child. THey didnt see it though. The ones that developed MS, had paranodal proteins (DCC, UNC5B, neurofascin). • Compact myelin presents a challenge for intracellular traffic. • How do organelles and proteins that are made in the cell body access the paranodal loops? Compact myelin Axon Omar de Faria Jr. How does the cell body communicate with and support the paranodal loops and the compact myelin in mature CNS myelin? Myelin unrolled, revealing the distribution of membrane associated with compact myelin and the organization of paranodal memebrane outer channel on the top that links cell body to paranodal loops paranodal loops Inner channel that runs inside paranodal loops these structures are huge! IN schwann cells have Schmidt landerman incisors for stability but what do oligodend. have ? node of Ranvier Consider if might mature myelin have the capacity to reorganize and regenerate. Plasticity? Non compacted PNS myelin: Paranodal loops, Cajal Bands and Schmidt-Lantermann Incisures Green - F-actin Cajal bands (illustrated) and S-L incisures are channels formed by non-compacted Schwann cell membranes. Cajal bands on surface, and S-L incisures forming a continuous channel that traverses the compact myelin, connecting the cell body cytoplasm with the paranodal loops (2004). a. drawing by Santiago Ramon y Cajal (1933) b. PNS nerve fiber stained with phalloidin (green) marking F-actin, and for DRP2 (red) marking compact myelin. c. Phalloidin staining in wild type mouse compared to mouse knockout for the Schwann cell protein periaxin. d. EM through PNS nerve fiber showing Cajal bands * e. EM showing junction of Cajal band and compact myelin. f. EM through PNS nerve fiber of periaxin ko mouse. No Cajal bands. Loss of Periaxin in humans or mouse results in a loss of Cajal bands, reduces internode length, and slows the rate of conduction velocity. Ultimately results in a severe PNS demyelinating neuropathy. 2014: thin cytoplasmic channels described in developing CNS myelin in vivo that link the OL cell body to the leading edge of the myelin sheath. TOM20 (mitochondria) MBP (myelin) Myelin sheath trying to myelinate the bottom of the dish TOM20 MBP Snaidero et al. 2014, Cell, M Simons lab Diane Nakamura Even mitochondria enter and migrate through channels within the myelin sheath to support myelin maintenance. Rinholm et al. 2016 (GLIA) Mitochondria 200nm you Dr.have Omar demitochondria Faria Jr. in paranodal loops Le Petit Prince, 1943 Mitochondrial migration is not restricted by the diameter of oligodendrocyte channels • Mitochondrial fusion/fission events are visible from outside the cell DIC DIC MitoTracker t = 0m 1um 1um t = 5m 1um MitoTracker dye t = 15m t = 20m mitochondria is bigger than the channel so there has to be fluidity to the channel Diameter (um) t = 10m 0.6 0.5 0.4 0.3 0.2 0.1 0 Mitochondria Nakamura et al 2020 GLIA How does the cell body communicate with and support the paranodal loops and the compact myelin in mature CNS myelin? Myelin unrolled, revealing the distribution of membrane associated with compact myelin and the organization of paranodal memebrane paranodal loops node of Ranvier Consider if might mature myelin have the capacity to reorganize and regenerate. Plasticity?