The Adult Oligodendrocyte Can Participate in Remyelination

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The adult oligodendrocyte can participate in remyelination

Ian D. Duncana,1, Abigail B. Radcliffa, Moones Heidaria, Grahame Kiddb, Benjamin K. Augustc, and Lauren A. Wierengaa

aDepartment of Medical Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, Madison, WI 53706; bRenovo Neural, Inc., Cleveland, OH 44106; and cElectron Microscope Facility, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, WI 53706

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved November 1, 2018 (received for review May 9, 2018) Endogenous remyelination of the CNS can be robust and restore drocytes, if there is persuasive evidence that they are capable of function, yet in multiple sclerosis it becomes less complete with time. participating in myelin repair. Promoting remyelination is a major therapeutic goal, both to restore The case against adult oligodendrocytes comes from both function and to protect axons from degeneration. Remyelination is in vivo and in vitro data. Blakemore and coworkers (17) have thought to depend on oligodendrocyte progenitor cells, giving shown that human oligodendrocytes transplanted into demyeli- rise to nascent remyelinating oligodendrocytes. Surviving, ma- nated areas of the rat spinal cord survived but were unable to ture oligodendrocytes are largely regarded as being uninvolved. remyelinate. Similarly, surviving endogenous postmitotic oligo- We have examined this question using two large animal models. dendrocytes did not remyelinate axons in areas of demyelination In the first model, there is extensive demyelination and remyelina- created by injection of antibody to Galactocerebroside (18). In tion of the CNS, yet oligodendrocytes survive, and in recovered vitro studies in which select stages of the oligodendrocyte lineage animals there is a mix of remyelinated axons interspersed between were added to cultures of retinal ganglion cells reported that only mature, thick myelin sheaths. Using 2D and 3D light and electron perinatal and adult OPCs developed into myelinating oligoden- microscopy, we show that many oligodendrocytes are connected to drocytes (19). Most recently, fate-mapping experiments reported mature and remyelinated myelin sheaths, which we conclude are that mature oligodendrocytes took no part in the remyelination of cells that have reextended processes to contact demyelinated axons focal, lysolecithin-induced demyelination in the mouse spinal cord while maintaining mature myelin internodes. In the second model (20). However, none of these data exclude the potential role of in vitamin B12-deficient nonhuman primates, we demonstrate that adult oligodendrocytes that lose some of their internodes follow- NEUROSCIENCE surviving mature oligodendrocytes extend processes and ensheath ing primary attack on myelin sheaths, yet remain intact and viable. demyelinated axons. These data indicate that mature oligodendro- The case for the possible role of the adult oligodendrocyte in cytes can participate in remyelination. remyelination is less well documented yet requires consideration. Initially it was proposed that mature oligodendrocytes could adult oligodendrocyte | remyelination | large animal models proliferate and provide more oligodendrocytes for remyelination (21). In vitro studies by Wood and Bunge (22) suggested that emyelination is the most effective and robust endogenous cell-sorted mature oligodendrocytes were more capable of gen- Rrepair mechanism of the central nervous system (CNS) and erating myelinating oligodendrocytes in cocultures than OPCs. can lead to complete remyelination of large and disseminated Transplantation of similarly sorted mature oligodendrocytes, areas of demyelination in the CNS, with resultant functional showed that they were also capable of myelinating axons in vivo recovery (1, 2). It has been known for some time that remyeli- (23, 24). In vitro studies of mature oligodendrocytes subjected to nation occurs in multiple sclerosis (MS) (3–5), although it has process disruption by various methods, have shown that these been reported as being limited, variable in patient populations, cells may demonstrate plasticity, regrow processes, and are ca- and more robust in early rather than late disease (6). There has pable of ensheathing and remyelinating axons. Oligodendrocyte been both extensive research and speculation into why this is so (2). Two detailed studies in 2006–2007 analyzed remyelination in Significance the forebrain of MS patients with different disease course and duration (7, 8). Perhaps surprisingly, extensive—albeit variable— Remyelination of the CNS is a critical process in restoring func- remyelination was seen in older patients, some of whom had long- tion and protecting nerve fibers from degeneration in multiple standing MS. Although it was not known when remyelination sclerosis and other demyelinating diseases. It is currently thought occurred in these patients, these observations underscore the ability that myelin can only be repaired by the generation of new oli- of even the mature brain in long-lasting MS to remyelinate or godendrocytes from progenitor cells and that remaining mature sustain earlier myelin repair. However, the lack of complete en- cellsareunabletoparticipate.Hereweshow,usinguniquelarge dogenous response has led to remyelination becoming a major animal models, including a nonhuman primate, that oligoden- therapeutic target, both to restore function and as the ultimate drocytes that are partially injured can participate in myelin re- form of neuroprotection (9–13). pair. The capacity of mature oligodendrocytes to remyelinate in The origin of remyelinating oligodendrocytes in MS and in demyelinating disease remains unknown, yet it provides an ad- experimental models of demyelination has been the subject of ditional cell source for recruitment for myelin repair. considerable interest. The weight of available evidence suggests Author contributions: I.D.D. designed research; A.B.R., B.K.A., and L.A.W. performed re- that remyelinating oligodendrocytes arise from oligodendrocyte search; G.K. contributed new reagents/analytic tools; I.D.D., M.H., and G.K. analyzed data; progenitor cells (OPCs) that reside either in or adjacent to and I.D.D. wrote the paper. demyelinated lesions, or adult neural stem cells residing in the The authors declare no conflict of interest. subependymal zone (14, 15). Experimental proof that OPCs This article is a PNAS Direct Submission. generate remyelinating oligodendrocytes comes from many Published under the PNAS license. transplant studies in de- and dysmyelinating models, as well as 1To whom correspondence should be addressed. Email: [email protected]. from toxin-induced demyelinating disorders (15, 16). However, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. new strategies to promote remyelination should consider recruiting 1073/pnas.1808064115/-/DCSupplemental. all cells of the oligodendrocyte lineage, including adult oligoden-

www.pnas.org/cgi/doi/10.1073/pnas.1808064115 PNAS Latest Articles | 1of10 Downloaded by guest on September 28, 2021 processes disrupted by transection or exposure to NMDA, Examination for Cell Death in Acute or Chronic Disease. Apoptotic retracted back to the cell body but regrew within 36 h (25). cells, if present, can be readily detected in 1-μm plastic sections Reextension or recovery of processes of mature oligodendro- (31) but despite examining multiple sections at all levels of the cytes following damage has also been shown in vivo. In a model spinal cord, optic nerves and brain from 14 cats, few or no of focal cerebral ischemia, McIver, et al. (26) showed that some pyknotic nuclei were seen at any stage of the disease. Similarly, oligodendrocytes survived, although with fragmented processes. cells at the early stage of apoptosis with condensed chromatin Oligodendrocytes that had been prelabeled with eGFP were (31), or pyknotic cells, were rarely seen on ultrastructural anal- found 1 wk later in areas of ischemia with intact processes, yses during acute disease. To confirm these observations, we used the TUNEL assay on spinal cord tissue from four cats suggesting that they may be able to participate in white matter + during acute disease. In one affected cat, no TUNEL cells were repair (26). Similarly, mature oligodendrocytes at the edge of + demyelinated lesions in the spinal cord, that overexpressed observed. In the other three affected cats, only 3 to 12 TUNEL cells were counted in the whole spinal cord cross-section (SI ERK1/2, extended processes into the lesions and remyelinated Appendix C D + axons (27). , Fig. S1 and ). Several of the TUNEL cells found in the affected cat tissue were within myelin vacuoles (SI Ap- Despite these data, the dominant opinion remains that remyelination pendix D SI Ap- is dependent upon the OPC, yet it is critical to consider more , Fig. S1 ), suggesting they were macrophages ( pendix, Fig. S1D). The tissue sections from cats on the normal mature cells of the oligodendrocyte lineage if alternative models + diet did not contain any TUNEL cells (SI Appendix, Fig. S1A), provide the opportunity to reexamine this question. We have ex- while all positive control tissue sections showed many brown, plored this issue in two models. The first unique model is fe- darkly stained nuclei throughout the section (SI Appendix, line irradiated food-induced demyelination (FIDID), in which Fig. S1B). demyelinated and remyelinated axons are found mixed with mature myelin sheaths (1). Mature myelin sheaths are those that have a Oligodendrocytes Are Seen to Contact and Ensheath both Remyelinated + normal g-ratio (axon diameter/axon myelin sheath diameter) Axons and Those with Mature Myelin Sheaths. The lateral and (<0.75), while remyelinated axons have thin sheaths with g-ratios ventral columns of four cats that showed a mix of remyelinated of over 0.75, in contrast to demyelinated axons that lack any axons and mature myelin sheaths were examined in detail to myelin (g-ratio 1.0). From our data, we propose that surviving determine whether oligodendrocytes could be seen to extend adult oligodendrocytes in such lesions are primarily responsible processes to both thick and thin myelin sheaths. In the animal for the robust remyelination seen. We have also examined ar- where remyelination was complete 2 y after recovery (Fig. 1), the chived spinal cord tissue from a model of vitamin B12 deficiency lack of space in the neuropil between myelinated axons made in a rhesus monkey in which there was extensive demyelination this difficult to visualize. However, in the two other cats, which but oligodendrocyte survival, mimicking the neuropathology of were examined 2–3 mo after cessation of the irradiated diet, we subacute combined degeneration (28, 29). Here we found evi- found numerous examples where an oligodendrocyte cell body dence of adult oligodendrocytes initiating remyelination by was seen to extend processes to both mature myelin sheaths and reensheathing axons, and in some instances producing thin myelin thinly remyelinated axons, and there was direct continuity be- sheaths. These complementary models provide strong evidence that tween the oligodendrocyte membrane and the outer lamella of in certain pathological milieu, the adult oligodendrocyte can par- the myelin sheath (Fig. 2 A–F). These relationships were inde- ticipate in remyelination. pendent of axon size: that is, the mature/thin myelin sheaths arising from a single oligodendrocyte were seen around both large Results and small diameter axons. Many of these glial units were sur- Scattered Demyelination and Remyelination Lead to a Mix of rounded by astrocyte processes, with no other adjacent glial cell Remyelinated and Mature Myelin Sheaths in the Lateral and Ventral nuclei, suggesting their potential isolation from other oligoden- Columns but Severe Demyelination in the Dorsal Column. Six months drocytes. Confirmation that individual myelin sheaths were either after beginning the irradiated diet, cats developed progressive mature or new (remyelinated) was demonstrated by measuring the hind leg ataxia and weakness. At this point, there was extensive g-ratios in these oligodendrocyte/axon groups (Fig. 2). “ ” demyelination and concurrent remyelination of the spinal cord in The oligodendrocyte nuclei and cytoplasm within these glial units A B cats. In the ventral and lateral columns, scattered demyelinated were variable in appearance. Those in Fig. 2 and , had copious and remyelinated axons were mixed with mature myelin sheaths cytoplasm and organelles, suggesting that active remyelination had C–F around axons of all calibers (Fig. 1B). In contrast, in the dorsal occurred recently compared with those in Fig. 2 ,wherethe column, large, contiguous areas of demyelination were seen (Fig. cells looked more mature. To illustrate the frequency of oligo- 1D). With recovery, mature myelin sheaths (defined as those dendrocytes connected to both thick and thin myelin sheaths, we with a g-ratio of 0.75 or less) remained dispersed throughout the identified an additional 13 oligodendrocytes from two animals that had clear cytoplasmic association with each cell, and mea- ventral and lateral columns, both at early and complete stages of sured the g-ratios of fibers, as demonstrated in Fig. 2G.Each remyelination (Fig. 1C), suggesting that these myelin sheaths oligodendrocyte was connected to mature and thin myelin sheaths were unaffected by the demyelinating “insult.” Hence, their that had g-ratios ranging from 0.6 to 0.92, confirming that many oligodendrocytes had remained intact yet close to demyelinated oligodendrocytes had maintained mature myelin internodes while axons (Fig. 1). Around 40% of axons of all calibers had thin remyelinating adjacent demyelinated axons. myelin sheaths, as previously reported (1). The pattern seen on recovery, which is a mixture of mature and remyelinated myelin Serial EM Imaging of Oligodendrocyte Ensheathment of Mature and sheaths, persisted over time (30). In one animal examined 2.5 y Thin Myelin Sheaths. The cellular connections underpinning after termination of the irradiated diet, the pattern of mixed remyelination were investigated using automated serial electron distribution of remyelinated and mature myelin sheaths remained microscopy (EM) imaging. In eight image sets, 44 partial or in the ventral column (Fig. 1). Examination of longitudinal sec- complete oligodendrocytes were identified, including 6 with tions through these areas of white matter showed that there were well-defined connections to both mature and relatively thin many short internodes on thinly myelinated (remyelinated) axons myelin. Three examples are shown in Fig. 3 A–F (OL1), Fig. 3 (30). In contrast to the lateral and ventral column, remyelination G–N (OL2), and Fig. 3 O–Q (OL3). Characteristic of some of the dorsal column resulted in large areas of homogeneously mature oligodendrocytes, OL1 (Fig. 3 A–F, overlaid in green) thin myelin sheaths (Fig. 1E). featured a prominent trunk-like process that was rich in

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808064115 Duncan et al. Downloaded by guest on September 28, 2021 microtubules and extended for at least 75 μm. Adjacent to the cell body, OL1 surrounded a short internode (Fig. 3 A–C,Ax1) that consisted of four to five myelin wraps and was only 44-μm long. Reconstruction of serial images confirmed direct OL1 cell body contact with the axon (Fig. 3B, green) and that OL1 contributed to paranodal loops (Fig. 3B, spiral loops in yellow). Somatic plasma membrane also formed the outer membrane of the mesaxon (Fig. 3C, arrow), which is the most lateral component of the spiral myelin membrane. A mature fiber (Fig. 3A, Ax3) was also connected at the cell soma (detail not shown). The OL1 trunk process gave rise to a fine cytoplasmic process (Fig. 3 D and E, yellow) that connected with a small axon (Fig. 3 A and D–F, Ax2) and extended a single-layer myelin wrap for 5 μm along the axon surface. This single wrap featured a mesaxon (Fig. 3E, arrow), partial com- paction, and had a g-ratio > 0.90, indicating that it was an early stage of remyelination. A nearby mature myelin internode (g-ratio 0.61) also connected with the main process (Fig. 3 A and F, Ax3; arrow, mesaxon, yellow outer tongue process). It should be noted that oligodendrocyte processes connecting with short, thin, newly formed internodes contained abundant cytoplasm, whereas connections to mature (low g-ratio) internodes were typically narrow with condensed cytoplasm. Because these latter were more difficult to follow with confi- dence, it is likely many connections to mature fibers, such as those surrounding OL1 (Fig. 3A), were overlooked. OL2 (Fig. 3G) lay close to OL1, and also surrounded both thin (Fig. 3 G and H, Ax1) and thick internodes (Fig. 3 G and I, Ax2) around NEUROSCIENCE the margins of the cell body. On one thick fiber (Fig. 3I,Ax2), an oligodendrocyte process (Fig. 3I, asterisk and yellow), extended around the myelin circumference to form the outer mesaxon (Fig. 3I, arrow). Distal to the cell body, the primary process (green) contacted several axons (Fig. 3J,Ax3–5). Dig- ital reslicing (Fig. 3J) demonstrated contacts with a high g-ratio internode (Fig. 3J, Ax3), the node of another (Fig. 3J,Ax4),and a low g-ratio fiber (Fig. 3J, Ax5). In serial images of Ax4 (Fig. 3 K–M), a branch of the nodal OL2 process was followed into paranodal processes (Fig. 3L, yellow) of a thin internodal myelin (Fig. 3M, Ax4). Contact between OL2 and a 5-μm diameter axon (Fig. 3 G and N, Ax6; g-ratio 0.65) suggested possible connection to that internode also. OL3 (Fig. 3O, green) encircled a large myelinated fiber (Fig. 3O, Ax1) (7 μmaxon,g- ratio0.59)andformedamesaxon(Fig.3P) as the outermost tongue process (Fig. 3P, green). OL3 also extended a process for 70 μmtoassociatewithasmallaxon(Fig.3O and Q,Ax2), where it encircled the axolemma at the node (Fig. 3Q,yellow, paranodal loops) and formed the myelin internode. Note that both oligodendrocytes 2 and 3 had other branches that also myelinated additional thin and thick myelin internodes.

Demyelination and Remyelination in a Nonhuman Primate Model. As previously reported, 3 y of vitamin B12 deprivation led to the development of large areas of demyelination in the spinal cord, with marked distension of the extracellular space allowing the visualization of individual oligodendrocytes and their processes (28, 29) (Fig. 4). At the edge of these lesions, some oligodendrocytes were noted to have connections only to mature myelin sheaths (Fig. 4B) or were adjacent to scattered demyelinated

Fig. 1. Mature myelin sheaths are seen in ventral columns at all stages of shown in Inset). At this stage, occasional demyelinated and remyelinated the disease and are mixed with demyelinated and remyelinated axons while axons are present. (C) In the ventral column of the recovered animal, there is the dorsal column initially has large areas of demyelination. (A) Normal a mix of remyelinated and mature myelin sheaths around axons of all di- mature white matter in the feline spinal cord ventral column. There is a mix ameters. (D) In the subpial area of the dorsal column of the same animal in B, of large, medium, and small diameter axons with appropriately thick myelin there is complete demyelination with myelin debris. The axons are much sheaths. (B) At the onset of disease in the ventral column, many axons be- smaller than in B as they are part of the fasciculus gracilis. (E) In the dorsal come vacuolated, yet in each the axon remains intact (arrows). As myelin column of the same animal shown in C, all of the axons have thin remyeli- breaks down, intact axons can still be seen (arrowhead and high power nated sheaths, different from the mosaic shown in C. (Scale bars, 20 μm.)

Duncan et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 28, 2021 Fig. 2. Oligodendrocytes have cytoplasmic connections to axons of variable diameter with thin or thick myelin sheaths. These five oligodendrocytes have cytoplasmic connections, seen on high power, contacting axons that have mature sheaths (g-ratios < 0.75) and thin myelin (remyelinated) sheaths (g-ratio > 0.75). Remyelinated myelin sheaths were seen around small diameter (A–C and E) or large diameter (D)axons.(F) Definitive cytoplasmic connection is seen in an oligodendrocyte that myelinates a mature myelin sheath (g-ratio 0.60) and remyelinated axon (g-ratio 0.84). Insets confirm the sheaths are derived from this cell (arrows). (G) Histogram detailing the oligodendrocyte/myelin sheath connections of 17 oligodendrocytes derived from two animals. The g-ratios were measured for each myelin sheath with which they had connections. The variability in each demonstrates that many mature oligodendrocytes have participated in remyelination. [Scale bars: 1 μm(A, C, E, and F), 2 μm(D), and 200 nm (F, Inset).]

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Fig. 3. SBC–EM images of three oligodendrocytes from cat ventral spinal cord during recovery, illustrating connection to developing and mature myelin internodes. Numbers throughout indicate g-ratios. (A) Oligodendrocyte (OL1 green) digitally resliced to show the cell body, principal process, and connected myelin on axons (Ax1–Ax3). (B and C) Two levels of Ax1. One process (B, green) at paranodal loops (B, yellow) was followed directly to cell body as the outermost cytoplasmic compartment of the outer tongue process. (C) At mid-cell body level (12 μm deeper than B), the plasma membrane of the cell body (green) forms the outer mesaxon (arrow) against the inner aspect of the outer tongue process (yellow). Myelin is thickest here (g-ratio 0.87). (D and E) Thin process (yellow) extends from trunk and encircles an axon (Ax2). Digital reslice image (D) shows continuity of process to axon, and in E, process (yellow) encircles the axon once and forms the mesaxon (arrow). (F) Trunk process (green) also forms mesaxon (arrow) with the outer tongue (yellow) of low g-ratio internode (Ax3). (G) Oligodendrocyte (OL2 green) digitally resliced to follow one major process and connecting axons (Ax1–Ax6). (H and I) The somatic membrane surrounds a small fiber (H; Ax1, axon) and thick myelin (I; axon Ax2). The process indicated by an asterisk encircles the internode perimeter to become an inner aspect of the outer tongue (yellow) at the mesaxon (yellow). (J) Digital reslice image following process of OL2 (green) which forms high g-ratio internode (Ax3, axon; yellow, outer tongue), contacts the surface of the adjacent axon (Ax4) and encircles a thick myelin internode (Ax5, axon). (K–M) Three planes of axon (Ax4). At the node (K), OL2 process (green) contacts axolemma, to become paranodal loops (L, yellow), and produce the thin internode (M, thickest myelin). (N) The somatic membrane also contacts surface of thicker myelin internode (Ax6, axon), suggesting possible connection. (O) Oligo- dendrocyte (OL3, green) digitally resliced as for G. The process encircles large axon (Ax1) and myelin, and forms a mesaxon (P, arrow) at the outer tongue (yellow). Another process extends to surround the nodal axon (Q, Ax2) and form paranodal loops (yellow) and myelin. OL over oligodendrocyte nucleus in A and G. [Scale bars: 1 μm(B–F, H–N, P, and Q) and 5 μm(A, G, and O).]

Duncan et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 28, 2021 Fig. 4. Vitamin B12 deficiency in the rhesus macaque is primarily a demyelinating disease. (A) Focal area of myelin vacuolation in the dorsal (posterior) column with pronounced increase in the intracellular space. (B) At the edge of the lesion adult oligodendrocytes myelinating normal-thickness myelin sheaths (*) were seen (g-ratios noted). (C) Scattered demyelinated axons were also seen at the edge (arrow). (D) Within the core of the lesion, most myelin sheaths were vacuolated but axons were intact (arrows). Demyelinated axons were present in the neuropil (arrowheads). (E) These were confirmed on EM. [Scale bars: 4 μm(E), 10 μm(B), 20 μm(C and D), and 200 μm(A).]

axons (Fig. 4C). In the core of these lesions there was extensive consider whether there are alternative explanations of oligo- myelin vacuolation leading to widespread demyelination (Fig. 4 dendrocytes that are connected to asymmetrically thick and thin D and E). In response to the myelin damage and demyelination, myelin sheaths. One potential explanation is that this finding oligodendrocytes both adjacent to and within the lesion dem- results from asynchronous development of myelin sheaths during onstrated a wide array of responses (Fig. 5). These oligoden- remyelination by newly generated oligodendrocytes. However, in drocytes were frequently associated with mature myelin sheaths development myelination of the ventral column of the cat spinal and had processes that were contacting, ensheathing, and in cord (the same species and area examined in this study) has been some instances, remyelinating demyelinated axons (Fig. 5). analyzed in detail by Remahl and Hildebrand (32). Using serial Remyelination was not as frequent as in FIDID, yet both thinly sectioning EM, the authors concluded that myelin sheaths as- remyelinated axons and those with short internodes were seen sociated with single oligodendrocytes had similar myelin sheath (Fig. 5). Ultrastructural analysis confirmed that oligodendrocytes thickness. Whether this is true in the adult CNS should be that had associated mature myelin sheaths also had demyeli- considered, given the growing interest and description of plas- nated axons embedded within their cytoplasm, as the first step ticity in mature white matter (33, 34). It is clear that myelin toward remyelination (Fig. 6). remodeling occurs in the adult brain, perhaps associated with ongoing oligodendrogenesis. However, in both rodents (35) and Discussion humans (36), the data suggest that there is little turnover of We present compelling evidence in two models of demyelination oligodendrocytes; hence, myelin remodeling would result from that adult oligodendrocytes can play a role in remyelination in preexisting oligodendrocytes (37). Such myelin sheath changes the spinal cord. This conclusion is based on 2D and 3D analyses would likely not be comparable to the large-scale remyelination of oligodendrocytes with cytoplasmic connections to both thick, seen in FIDID and other demyelinating diseases. In FIDID we mature myelin sheaths and thin remyelinated sheaths. Our in- have shown that the mosaic pattern of myelin sheaths persists for terpretation of this finding is that such oligodendrocytes had over 2 y with no long-term remodeling (30). survived the myelin breakdown, maintaining one or more of their The alternative hypothesis that asynchronous myelin sheath original internodes while reextending processes to remyelinate thickness is a feature seen on remyelination but not in development adjacent demyelinated axons (Fig. 7). However, it is critical to should also be considered. However, review of the remyelinating

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Fig. 5. Oligodendrocytes respond to demyelination by contacting naked axons and engulfing them in their cytoplasm, resulting in restricted remyelination. In this montage, a range of the early and late interactions of mature oligodendrocytes with demyelinated axons is seen. Early contact with demyelinated axons is seen in A–F and H, with fine processes (arrows) beginning to contact and surround axons (denoted by “a”). The adult oligodendrocyte in B is connected to two axons with mature myelin sheaths (*, g-ratios 0.75 and 0.67) and has a process connected to a demyelinated axon (a). Demyelinated axons can be seen indented within the oligodendrocyte cytoplasm (D) and within oligodendrocytes that have both mature and remyelinated sheaths (E). A mature oligodendrocyte myelinates both thin (*, g = 0.89) and thick (*, g = 0.55) sheaths (F). Remyelination is confirmed by the presence of short, thin internodes (G). [Scale bars: 5 μm(C, Inset), 10 μm(A, B,andD–H), and 20 μm(C).]

pattern in a range of species following demyelination in toxic While studies of several models of remyelination have concluded diseases—including cuprizone, lysolecithin and ethidium bromide that mature oligodendrocyte are incapable of remyelination (2), it is (38–44), multiple sclerosis (45, 46), and viral demyelinating dis- important to note that the two models presented here are different ease (47, 48)—shows the generation of uniformly thin myelin from the in vitro and in vivo models used previously to define the sheaths. In addition, transplantation of neonatal and adult glial OPC as the primary, if not the sole, origin of remyelinating oligo- cells from different species into de- or dysmyelinated lesions, also dendrocytes in many experimental systems. Therefore, it may be results in large, confluent areas of thinly myelinated axons (49– that the lesion type dictates the cellular response. In large yet focal 53). Hence we conclude that in the model presented here, the areas of demyelination in which there is marked oligodendrocyte most likely interpretation of an oligodendrocyte connected to loss, such as in some MS plaques, remaining or adjacent OPCs may mature and remyelinated myelin sheaths is that it is a mature be the only or primary source of remyelinating oligodendrocytes. In oligodendrocyte that has participated in remyelination. lesions, such as seen in FIDID or vitamin B12 deficiency, where

Duncan et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 28, 2021 oligodendrocytes that are scattered throughout the demyelinated milieu may respond promptly with process extension (Fig. 7), as occurs in vitro within 4 h of process damage (25). Whether there is organelle redistribution in such processes that promote the generation and transport of myelin proteins or their RNAs, including Golgi and microtubules, will require further investigation. Therefore, if the adult oligodendrocyte might be a player in remyelination, does this have significance in MS and other human myelin disorders? We have proposed here that surviving adult oligodendrocytes are attached to at least a single internode to be viable. While oligodendrocyte death is a significant finding in MS at different stages of the disease, surviving mature oligo- Fig. 6. EM confirms the oligodendrocyte/demyelinated axon interaction. (A dendrocytes, although rare in chronic lesions (61), can be present and B) Two examples of oligodendrocytes connected to mature myelin and occasionally in large numbers (62–64). MS plaques most sheaths that have initiated early ensheathment of demyelinated axons (denoted by “a”). (Scale bars, 2 μm.) often consist of large, contiguous areas of demyelinated axons; hence, surviving oligodendrocytes in the middle of such plaques may be incapable of process reextension, as they have no intact demyelinated axons are scattered among persistent mature mye- internodes. Although OPCs may be the only cell capable of linated axons and there is little or no oligodendrocyte death, sur- generating remyelinating oligodendrocytes in such lesions, it has viving mature oligodendrocytes may be recruited to take part in previously been suggested that surviving, mature oligodendro- myelin repair. cytes may be involved (62). Additionally, at the periphery of The critical difference between the models we describe here plaques, intact oligodendrocytes may have lost some internodes and the diverse in vitro and in vivo studies on the cellular basis of but not all, and may extend processes into the plaque. In FIDID, remyelination is that the adult oligodendrocytes are intact and the extent of demyelination varies depending on the anatomical viable as they retained an intact internode. Transplantation of site: that is, in the lateral and ventral columns of the spinal cord, mature oligodendrocytes into the demyelinated spinal cord (54) it is patchy, whereas in the dorsal column and optic nerve it is or plating them in cocultures (19) could challenge oligodendro- much more generalized. In the optic nerve, which can totally cytes to reinitiate the entire temporal sequence of events that demyelinate, remyelination can be complete and thus OPCs may occur in development. Such cells may have to de-differentiate, be the cell or origin of remyelinating oligodendrocytes in this divide, then express the genes required to generate the myelin structure and in the dorsal column, leading to predominately thin sheath in the correct temporal sequence. The study of Makinodan myelin sheaths (Fig. 1E), in contrast to the rest of the spinal cord et al. (25) showed no evidence of division of oligodendrocytes in (Fig. 1C). Therefore, in the same disorder, the OPC and mature which their processes were damaged, even after the addition of oligodendrocyte may play complimentary, yet CNS site-specific PDGF, hence the reextension of processes did not require the cell roles in myelin repair. to divide or de-differentiate. Other experimental studies that have The observations of mature oligodendrocytes in the primate argued against the mature oligodendrocytes used strategies, such model initiating the process of reensheathment and myelination as irradiation of the CNS or transplantation that could have a of demyelinated axons provides strong support that this may negative effect on the remyelinating ability of these cells (20). occur in MS lesions. In the rhesus macaque lesions, identification The strongest data supporting the limited functional capacity of single oligodendrocytes and their processes is aided by the of mature oligodendrocytes as sources of myelin repair comes increase in the extracellular space in lesions (29). This model from studies of myelination of the zebrafish spinal cord (55). mimics subacute combined degeneration (SCD) that results from Using live imaging, they showed that oligodendrocytes have a vitamin B12 deficiency (60, 65). The myelopathy that develops in very brief window of time (5 h) during development in which to SCD may be the “purest” demyelinating disease of humans, and generate myelin sheaths and do so in a brief, synchronous neurological and radiological recovery on B12 treatment, likely fashion. Similar developmental limitations were seen in mam- occurs from remyelination, possibly resulting from persisting, malian coculture systems (19). In the studies reported herein, mature oligodendrocytes. The data presented here demonstrate however, the myelin breakdown, macrophage infiltration, and that surviving adult oligodendrocytes have the propensity to in- associated cytokine expression will create a very different envi- teract with demyelinated axons, initiate the wrapping and em- ronment from that seen during development. Similarly, surviving bedding of axons in oligodendrocyte cytoplasm, and in some oligodendrocytes abut demyelinated axons and therefore could instances, remyelinate these axons. While remyelination by adult enhance their remyelinating potential. oligodendrocytes may be less robust than in FIDID, the lack of What are the differences between the models reported here vitamin B12 will undoubtedly diminish the function of the oli- and those commonly used in studies on remyelination, which godendrocyte. Indeed, deprivation of B12 in the human brain may be relevant to the discussion on the cellular origin of myelin repair? Cuprizone, which is currently the de facto model of demyelination/remyelination, is very different as the toxin kills adult oligodendrocytes (56). Hence, remyelination after cessation of cuprizone treatment requires the generation of new oligodendrocytes from OPCs. Remyelinated axons have uniformly thin myelin sheaths (40, 57) that persist for over 6 mo (58, 59), resembling that seen in the dorsal column of cats with FIDID (Fig. 1E). In FIDID and also in B12 deficiency in the monkey and humans (60), there is no oligodendrocyte death. Importantly, at Fig. 7. Cartoon illustrating the proposed response of mature oligoden- least in FIDID in the lateral and ventral columns of the spinal drocytes to adjacent demyelinated internodes. There is partial loss of in- cord, remyelination occurs concurrently with demyelination; ternodes of this oligodendrocytes territory and retraction of processes. This hence, it would seem unlikely that there is time for OPCs to is quickly followed by process reextension and remyelination, resulting in divide, differentiate, and reensheath axons. In contrast, surviving thin, short internodes, while the axon maintains its original myelin sheaths.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808064115 Duncan et al. Downloaded by guest on September 28, 2021 during development results in severe myelination delay which, Rhesus Monkey Vitamin B12 Deprivation. Spinal cord tissue from a rhesus like SCD, can respond to B12 therapy with restoration of function macaque that was part of an experimental group of animals that had been of preexisting oligodendrocytes with myelin repair (66). fed a vitamin B12-deficient diet for over 3 y, and which had developed visual loss and lower limb weakness and ataxia, was examined by light and EM (28, Materials and Methods 29) (these studies were performed prior to University Animal Care Com- All cats were handled and treated according to the guidelines of the Research mittee oversight). The spinal cord tissue was removed from a monkey per- Animal Resources Center and the Animal Care and Use Committee at the fused with aldehyde fixative after being killed. Tissue was sectioned for light University of Wisconsin–Madison. The disorder (FIDID) was produced by microscopy and EM as above. feeding cats dried food irradiated at 45–55 kGy (Sterigenics Radiation Fa- – cility) (1). Cats developed progressive hind limb ataxia and paresis after 5 SBF–EM Imaging and Analysis of Spinal Cord Sections from Cats with FIDID. 6 mo on this diet. Return to a normal diet led to a slow recovery in all cats Tissues for SBF–SEM were fixed as above in modified Karnovsky fixative and but not those that had developed urinary incontinence. The cervical and stained and imaged by Renovo Neural Inc. Briefly, the samples were washed, thoracic spinal cord was collected from cats with FIDID both during active stained with 1% tannic acid for 30 min and then incubated successively with disease and partial or full recovery, 2–3 mo or 2 y after removal from the irradiated diet. Cats were perfused with a modified Karnovsky fixative and osmium ferricyanide, tetracarbohydrazide, osmium tetroxide, uranyl ace- segments of the spinal cord processed and embedded in plastic for light tate, and lead aspartate (67, 68). Tissues were dehydrated and embedded in microscopy, transmission electron microscopy (TEM), and serial blockface Epon-812 resin (Procure; Electron Microscopy Sciences). scanning–EM (SBF–SEM). One-micrometer sections were used to identify Samples were imaged using a Zeiss Sigma VP scanning EM equipped with a areas of remyelination and thin sections then cut for TEM. TEM was per- Gatan 3View in-chamber ultramicrotome stage with low-kilovolt back- formed on a Philips CM120 transmission electron microscope. Images were scattered electron detectors optimized for 3View systems. Stacks of digital captured with a BioSprint 12 series digital camera using AMT Image Capture images were acquired at 7–8 nm per pixel resolution (x,y) at 2.25 kV, using Engine V700. The g-ratios were measured on electron micrographs of myelin 80-nm steps; these are standard settings that approximate TEM-based sheaths that were seen to be adjacent to oligodendrocyte nuclei. Mature resulting serial image stacks contained 300–900 images (∼120 × 100 μm, myelin sheaths were defined as those with a g-ratio of 0.75 or less. 16,000 × 12,800 pixels each, 100–180 GB per set). For analysis, images were scaled, substacks generated, and aligned using ImageJ software with FIJI TUNEL Assay. TUNEL assay was performed to detect glial cell apoptosis. Following fixation, cervical spinal cord tissue from four affected cats and one plug-in suite. In some cases, cytoplasmic process continuity was illustrated by normal cat were embedded in paraffin. Paraffin blocks were then sectioned digitally reslicing the stack such that the visible plane followed the center- (4-μm thickness), deparaffinized, and rehydrated by the University of Wis- line of the process. Three-dimensional models were generated by manual consin–Madison School of Veterinary Medicine Histology Laboratory. Two tracing using Reconstruct software (69). NEUROSCIENCE cervical spinal cord sections were used per treatment group (positive control, negative control, and experimental TUNEL group) per animal, except for one ACKNOWLEDGMENTS. We thank Dr. Dimitri Agamanolis for his generous affected cat for which there was only a negative and experimental treat- donation of tissue blocks from his previous studies; Dr. Joshua Mayer for ment with one section/treatment group. Slides were stored in PBS overnight creating the cartoon used in Fig. 7; Emily Benson and Diandra Starks (Renovo) and then treated with the DeadEnd Colorimetric TUNEL Assay kit (Promega) for 3D electron microscopy imaging and tracing expertise; Drs. Jayshree according to the manufacturer’s protocol. Stained sections were examined Samanta and John Svaren for their comments on this manuscript; and Amin using a Nikon Eclipse E800 microscope equipped with a Nikon Digital Sight Sherafat for useful discussion. These studies were supported by National Mul- DS-Ri1 camera and NIS-Elements Documentation software to obtain images. tiple Sclerosis Society Grant RG-1501-02876.

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