Targeted Complement Inhibition at Synapses Prevents Microglial Synaptic Engulfment and Synapse Loss in Demyelinating Disease.

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.

Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

Mixed Lineage Kinase Domain-like Protein MLKL Breaks Down Myelin following Nerve Injury.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.

Dissection of complement and Fc-receptor-mediated pathomechanisms of autoantibodies to myelin oligodendrocyte glycoprotein.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have recently been established to define a new disease entity, MOG-antibody-associated disease (MOGAD), which is clinically overlapping with multiple sclerosis. MOG-specific antibodies (Abs) from patients are pathogenic, but the precise effector mechanisms are currently still unknown and no therapy is approved for MOGAD. Here, we determined the contributions of complement and Fc-receptor (FcR)-mediated effects in the pathogenicity of MOG-Abs. Starting from a recombinant anti-MOG (mAb) with human IgG1 Fc, we established MOG-specific mutant mAbs with differential FcR and C1q binding. We then applied selected mutants of this MOG-mAb in two animal models of experimental autoimmune encephalomyelitis. First, we found MOG-mAb-induced demyelination was mediated by both complement and FcRs about equally. Second, we found that MOG-Abs enhanced activation of cognate MOG-specific T cells in the central nervous system (CNS), which was dependent on FcR-, but not C1q-binding. The identification of complement-dependent and -independent pathomechanisms of MOG-Abs has implications for therapeutic strategies in MOGAD.

Staged suppression of microglial autophagy facilitates regeneration in CNS demyelination by enhancing the production of linoleic acid.

Microglia play a critical role in the clearance of myelin debris, thereby ensuring functional recovery from neural injury. Here, using mouse model of demyelination following two-point LPC injection, we show that the microglial autophagic-lysosomal pathway becomes overactivated in response to severe demyelination, leading to lipid droplet accumulation and a dysfunctional and pro-inflammatory microglial state, and finally failed myelin debris clearance and spatial learning deficits. Data from genetic approaches and pharmacological modulations, via microglial Atg5 deficient mice and intraventricular BAF A1 administration, respectively, demonstrate that staged suppression of excessive autophagic-lysosomal activation in microglia, but not sustained inhibition, results in better myelin debris degradation and exerts protective effects against demyelination. Combined multi-omics results in vitro further showed that enhanced lipid metabolism, especially the activation of the linoleic acid pathway, underlies this protective effect. Supplementation with conjugated linoleic acid (CLA), both in vivo and in vitro, could mimic these effects, including attenuating inflammation and restoring microglial pro-regenerative properties, finally resulting in better recovery from demyelination injuries and improved spatial learning function, by activating the peroxisome proliferator-activated receptor (PPAR-γ) pathway. Therefore, we propose that pharmacological inhibition targeting microglial autophagic-lysosomal overactivation or supplementation with CLA could represent a potential therapeutic strategy in demyelinated disorders.

Oligodendrocyte Maturation Alters the Cell Death Mechanisms That Cause Demyelination.

Myelinating oligodendrocytes die in human disease and early in aging. Despite this, the mechanisms that underly oligodendrocyte death are not resolved and it is also not clear whether these mechanisms change as oligodendrocyte lineage cells are undergoing differentiation and maturation. Here, we used a combination of intravital imaging, single-cell ablation, and cuprizone-mediated demyelination, in both female and male mice, to discover that oligodendrocyte maturation dictates the dynamics and mechanisms of cell death. After single-cell phototoxic damage, oligodendrocyte precursor cells underwent programmed cell death within hours, differentiating oligodendrocytes died over several days, while mature oligodendrocytes took weeks to die. Importantly cells at each maturation stage all eventually died but did so with drastically different temporal dynamics and morphological features. Consistent with this, cuprizone treatment initiated a caspase-3-dependent form of rapid cell death in differentiating oligodendrocytes, while mature oligodendrocytes never activated this executioner caspase. Instead, mature oligodendrocytes exhibited delayed cell death which was marked by DNA damage and disruption in poly-ADP-ribose subcellular localization. Thus, oligodendrocyte maturation plays a key role in determining the mechanism of death a cell undergoes in response to the same insult. This means that oligodendrocyte maturation is important to consider when designing strategies for preventing cell death and preserving myelin while also enhancing the survival of new oligodendrocytes in demyelinating conditions.

p38γ MAPK delays myelination and remyelination and is abundant in multiple sclerosis lesions.

Multiple sclerosis is a chronic inflammatory disease in which disability results from the disruption of myelin and axons. During the initial stages of the disease, injured myelin is replaced by mature myelinating oligodendrocytes that differentiate from oligodendrocyte precursor cells. However, myelin repair fails in secondary and chronic progressive stages of the disease and with ageing, as the environment becomes progressively more hostile. This may be attributable to inhibitory molecules in the multiple sclerosis environment including activation of the p38MAPK family of kinases. We explored oligodendrocyte precursor cell differentiation and myelin repair using animals with conditional ablation of p38MAPKγ from oligodendrocyte precursors. We found that p38γMAPK ablation accelerated oligodendrocyte precursor cell differentiation and myelination. This resulted in an increase in both the total number of oligodendrocytes and the migration of progenitors ex vivo and faster remyelination in the cuprizone model of demyelination/remyelination. Consistent with its role as an inhibitor of myelination, p38γMAPK was significantly downregulated as oligodendrocyte precursor cells matured into oligodendrocytes. Notably, p38γMAPK was enriched in multiple sclerosis lesions from patients. Oligodendrocyte progenitors expressed high levels of p38γMAPK in areas of failed remyelination but did not express detectable levels of p38γMAPK in areas where remyelination was apparent. Our data suggest that p38γ could be targeted to improve myelin repair in multiple sclerosis.

Deficiency of galactosyl-ceramidase in adult oligodendrocytes worsens disease severity during chronic experimental allergic encephalomyelitis.

Galactosyl-ceramidase (GALC) is a ubiquitous lysosomal enzyme crucial for the correct myelination of the mammalian nervous system during early postnatal development. However, the physiological consequence of GALC deficiency in the adult brain remains unknown. In this study, we found that mice with conditional ablation of GALC activity in post-myelinating oligodendrocytes were lethally sensitized when challenged with chronic experimental allergic encephalomyelitis (EAE), in contrast with the non-lethal dysmyelination observed in Galc-ablated mice without the EAE challenge. Mechanistically, we found strong inflammatory demyelination without remyelination and an impaired fusion of lysosomes and autophagosomes with accumulation of myelin debris after a transcription factor EB-dependent increase in the lysosomal autophagosome flux. These results indicate that the physiological impact of GALC deficiency is highly influenced by the cell context (oligodendroglial vs. global expression), the presence of inflammation, and the developmental time when it happens (pre-myelination vs. post-myelination). We conclude that Galc expression in adult oligodendrocytes is crucial for the maintenance of adult central myelin and to decrease vulnerability to additional demyelinating insults.

A novel in vitro model for investigating oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions: Impact of microglial depletion and repopulation.

Experimental models of multiple sclerosis (MS) have significantly contributed to our understanding of pathophysiology and the development of therapeutic interventions. Various in vivo animal models have successfully replicated key features of MS and associated pathophysiological processes, shedding light on the sequence of events leading to disease initiation, progression, and resolution. Nevertheless, these models often entail substantial costs and prolonged treatment periods. In contrast, in vitro models offer distinct advantages, including cost-effectiveness and precise control over experimental conditions, thereby facilitating more reproducible results. We have developed a novel in vitro model tailored to the study of oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions, which encompasses all the cell types present in the central nervous system (CNS). Of note, our model enables the evaluation of microglial cell commitment through a protocol involving their depletion and subsequent repopulation. Given that the development and survival of microglia are critically reliant on colony-stimulating factor-1 receptor (CSF-1R) signaling, we have employed CSF-1R inhibition to effectively deplete microglia. This versatile model holds promise for the assessment of potential therapies aimed at promoting oligodendroglial differentiation to safeguard and repair myelin, hence mitigate neurodegenerative processes.

Failed remyelination of the nonhuman primate optic nerve leads to axon degeneration, retinal damages, and visual dysfunction.

White matter disorders of the central nervous system (CNS), such as multiple sclerosis (MS), lead to failure of nerve conduction and long-lasting neurological disabilities affecting a variety of sensory and motor systems, including vision. While most disease-modifying therapies target the immune and inflammatory response, the promotion of remyelination has become a new therapeutic avenue to prevent neuronal degeneration and promote recovery. Most of these strategies have been developed in short-lived rodent models of demyelination, which spontaneously repair and do not reflect the size, organization, and biology of the human CNS. Thus, well-defined nonhuman primate models are required to efficiently advance therapeutic approaches for patients. Here, we followed the consequence of long-term toxin-induced demyelination of the macaque optic nerve on remyelination and axon preservation, as well as its impact on visual functions. Findings from oculomotor behavior, ophthalmic examination, electrophysiology, and retinal imaging indicate visual impairment involving the optic nerve and retina. These visual dysfunctions fully correlated at the anatomical level, with sustained optic nerve demyelination, axonal degeneration, and alterations of the inner retinal layers. This nonhuman primate model of chronic optic nerve demyelination associated with axonal degeneration and visual dysfunction, recapitulates several key features of MS lesions and should be instrumental in providing the missing link to translate emerging repair promyelinating/neuroprotective therapies to the clinic for myelin disorders, such as MS.

Remyelination-oriented clemastine treatment attenuates neuropathies of optic nerve and retina in glaucoma.

As one of the top causes of blindness worldwide, glaucoma leads to diverse optic neuropathies such as degeneration of retinal ganglion cells (RGCs). It is widely accepted that the level of intraocular pressure (IOP) is a major risk factor in human glaucoma, and reduction of IOP level is the principally most well-known method to prevent cell death of RGCs. However, clinical studies show that lowering IOP fails to prevent RGC degeneration in the progression of glaucoma. Thus, a comprehensive understanding of glaucoma pathological process is required for developing new therapeutic strategies. In this study, we provide functional and histological evidence showing that optic nerve defects occurred before retina damage in an ocular hypertension glaucoma mouse model, in which oligodendroglial lineage cells were responsible for the subsequent neuropathology. By treatment with clemastine, an Food and Drug Administration (FDA)-approved first-generation antihistamine medicine, we demonstrate that the optic nerve and retina damages were attenuated via promoting oligodendrocyte precursor cell (OPC) differentiation and enhancing remyelination. Taken together, our results reveal the timeline of the optic neuropathies in glaucoma and highlight the potential role of oligodendroglial lineage cells playing in its treatment. Clemastine may be used in future clinical applications for demyelination-associated glaucoma.

Microglia-derived extracellular vesicles in homeostasis and demyelination/remyelination processes.

Microglia (MG) play a crucial role as the predominant myeloid cells in the central nervous system and are commonly activated in multiple sclerosis. They perform essential functions under normal conditions, such as actively surveying the surrounding parenchyma, facilitating synaptic remodeling, engulfing dead cells and debris, and protecting the brain against infectious pathogens and harmful self-proteins. Extracellular vesicles (EVs) are diverse structures enclosed by a lipid bilayer that originate from intracellular endocytic trafficking or the plasma membrane. They are released by cells into the extracellular space and can be found in various bodily fluids. EVs have recently emerged as a communication mechanism between cells, enabling the transfer of functional proteins, lipids, different RNA species, and even fragments of DNA from donor cells. MG act as both source and recipient of EVs. Consequently, MG-derived EVs are involved in regulating synapse development and maintaining homeostasis. These EVs also directly influence astrocytes, significantly increasing the release of inflammatory cytokines like IL-1ß, IL-6, and TNF-α, resulting in a robust inflammatory response. Furthermore, EVs derived from inflammatory MG have been found to inhibit remyelination, whereas Evs produced by pro-regenerative MG effectively promote myelin repair. This review aims to provide an overview of the current understanding of MG-derived Evs, their impact on neighboring cells, and the cellular microenvironment in normal conditions and pathological states, specifically focusing on demyelination and remyelination processes.

Testosterone Inhibits Secretion of the Pro-Inflammatory Chemokine CXCL1 from Astrocytes.

Astrocytes play an important role in the regulation of the inflammatory response in the CNS, e.g., in demyelinating diseases. Since the chemokine CXCL1 is known to be secreted by astrocytes and to have a pro-inflammatory effect on immune cells in the CNS, we verified the effect of testosterone on its secretion in vitro (in the astrocytic cell line DI TNC1). Testosterone reduced the increase in CXCL1 production caused by the pro-inflammatory agent lysophosphatidylcholine and restored the basal production level of CXCL1. The androgen receptor (present and functional in the studied cell line) was strongly suggested to mediate this effect-its non-steroid ligand flutamide exerted an agonist-like effect, mimicking the activity of testosterone itself on CXCL1 secretion. This novel mechanism has important implications for the known immunomodulatory effect of testosterone and potentially other androgenic hormones. It provides a potential explanation on the molecular level and shows that astrocytes are important players in inflammatory homeostasis in the CNS and its hormonal regulation. Therefore, it suggests new directions for the development of the therapeutic intervention.

Cystatin F attenuates neuroinflammation and demyelination following murine coronavirus infection of the central nervous system.

BACKGROUND: Cystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination. METHODS: Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3' single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios. RESULTS: JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls. CONCLUSIONS: Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.

Arachidonic acid-derived lipid mediators in multiple sclerosis pathogenesis: fueling or dampening disease progression?

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by neuroinflammation, demyelination, and neurodegeneration. Considering the increasing prevalence among young adults worldwide and the disabling phenotype of the disease, a deeper understanding of the complexity of the disease pathogenesis is needed to ultimately improve diagnosis and personalize treatment opportunities. Recent findings suggest that bioactive lipid mediators (LM) derived from ω-3/-6 polyunsaturated fatty acids (PUFA), also termed eicosanoids, may contribute to MS pathogenesis. For example, disturbances in LM profiles and especially those derived from the ω-6 PUFA arachidonic acid (AA) have been reported in people with MS (PwMS), where they may contribute to the chronicity of neuroinflammatory processes. Moreover, we have previously shown that certain AA-derived LMs also associated with neurodegenerative processes in PwMS, suggesting that AA-derived LMs are involved in more pathological events than solely neuroinflammation. Yet, to date, a comprehensive overview of the contribution of these LMs to MS-associated pathological processes remains elusive. MAIN BODY: This review summarizes and critically evaluates the current body of literature on the eicosanoid biosynthetic pathway and its contribution to key pathological hallmarks of MS during different disease stages. Various parts of the eicosanoid pathway are highlighted, namely, the prostanoid, leukotriene, and hydroxyeicosatetraenoic acids (HETEs) biochemical routes that include specific enzymes of the cyclooxygenases (COXs) and lipoxygenases (LOX) families. In addition, cellular sources of LMs and their potential target cells based on receptor expression profiles will be discussed in the context of MS. Finally, we propose novel therapeutic approaches based on eicosanoid pathway and/or receptor modulation to ultimately target chronic neuroinflammation, demyelination and neurodegeneration in MS. SHORT CONCLUSION: The eicosanoid pathway is intrinsically linked to specific aspects of MS pathogenesis. Therefore, we propose that novel intervention strategies, with the aim of accurately modulating the eicosanoid pathway towards the biosynthesis of beneficial LMs, can potentially contribute to more patient- and MS subtype-specific treatment opportunities to combat MS.

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology.

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-ß-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine ß-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).

The CD40/CD40 ligand dyad and its downstream effector molecule ISG54 in relating acute neuroinflammation with persistent, progressive demyelination.

Although Multiple Sclerosis (MS) is primarily thought to be an autoimmune condition, its possible viral etiology must be taken into consideration. When mice are administered neurotropic viruses like mouse hepatitis virus MHV-A59, a murine coronavirus, or its isogenic recombinant strain RSA59, neuroinflammation along with demyelination are observed, which are some of the significant manifestations of MS. MHV-A59/RSA59 induced neuroinflammation is one of the best-studied experimental animal models to understand the viral-induced demyelination concurrent with axonal loss. In this experimental animal model, one of the major immune checkpoint regulators is the CD40-CD40L dyad, which helps in mediating both acute-innate, innate-adaptive, and chronic-adaptive immune responses. Hence, they are essential in reducing acute neuroinflammation and chronic progressive adaptive demyelination. While CD40 is expressed on antigen-presenting cells and endothelial cells, CD40L is expressed primarily on activated T cells and during severe inflammation on NK cells and mast cells. Experimental evidences revealed that genetic deficiency of both these proteins can lead to deleterious effects in an individual. On the other hand, interferon-stimulated genes (ISGs) possess potent antiviral properties and directly or indirectly alter acute neuroinflammation. In this review, we will discuss the role of an ISG, ISG54, and its tetratricopeptide repeat protein Ifit2; the genetic and experimental studies on the role of CD40 and CD40L in a virus-induced neuroinflammatory demyelination model.

Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

Automated whole slide morphometry of sural nerve biopsy using machine learning.

AIM: The morphometry of sural nerve biopsies, such as fibre diameter and myelin thickness, helps us understand the underlying mechanism of peripheral neuropathies. However, in current clinical practice, only a portion of the specimen is measured manually because of its labour-intensive nature. In this study, we aimed to develop a machine learning-based application that inputs a whole slide image (WSI) of the biopsied sural nerve and automatically performs morphometric analyses. METHODS: Our application consists of three supervised learning models: (1) nerve fascicle instance segmentation, (2) myelinated fibre detection and (3) myelin sheath segmentation. We fine-tuned these models using 86 toluidine blue-stained slides from various neuropathies and developed an open-source Python library. RESULTS: Performance evaluation showed (1) a mask average precision (AP) of 0.861 for fascicle segmentation, (2) box AP of 0.711 for fibre detection and (3) a mean intersection over union (mIoU) of 0.817 for myelin segmentation. Our software identified 323,298 nerve fibres and 782 fascicles in 70 WSIs. Small and large fibre populations were objectively determined based on clustering analysis. The demyelination group had large fibres with thinner myelin sheaths and higher g-ratios than the vasculitis group. The slope of the regression line from the scatter plots of the diameters and g-ratios was higher in the demyelination group than in the vasculitis group. CONCLUSION: We developed an application that performs whole slide morphometry of human biopsy samples. Our open-source software can be used by clinicians and pathologists without specific machine learning skills, which we expect will facilitate data-driven analysis of sural nerve biopsies for a more detailed understanding of these diseases.

The prevalence and topography of spinal cord demyelination in multiple sclerosis: a retrospective study.

Spinal cord pathology is a major determinant of irreversible disability in progressive multiple sclerosis. The demyelinated lesion is a cardinal feature. The well-characterised anatomy of the spinal cord and new analytic approaches allows the systematic study of lesion topography and its extent of inflammatory activity unveiling new insights into disease pathogenesis. We studied cervical, thoracic, and lumbar spinal cord tissue from 119 pathologically confirmed multiple sclerosis cases. Immunohistochemistry was used to detect demyelination (PLP) and classify lesional inflammatory activity (CD68). Prevalence and distribution of demyelination, staged by lesion activity, was determined and topographical maps were created to identify patterns of lesion prevalence and distribution using mixed models and permutation-based voxelwise analysis. 460 lesions were observed throughout the spinal cord with 76.5% of cases demonstrating at least 1 lesion. The cervical level was preferentially affected by lesions. 58.3% of lesions were inflammatory with 87.9% of cases harbouring at least 1 inflammatory lesion. Topographically, lesions consistently affected the dorsal and lateral columns with relative sparing of subpial areas in a distribution mirroring the vascular network. The presence of spinal cord lesions and the proportion of active lesions related strongly with clinical disease milestones, including time from onset to wheelchair and onset to death. We demonstrate that spinal cord demyelination is common, highly inflammatory, has a predilection for the cervical level, and relates to clinical disability. The topography of lesions in the dorsal and lateral columns and relative sparing of subpial areas points to a role of the vasculature in lesion pathogenesis, suggesting short-range cell infiltration from the blood and signaling molecules circulating in the perivascular space incite lesion development. These findings challenge the notion that end-stage progressive multiple sclerosis is 'burnt out' and an outside-in lesional gradient predominates in the spinal cord. Taken together, this study provides support for long-term targeting of inflammatory demyelination in the spinal cord and nominates vascular dysfunction as a potential target for new therapeutic approaches to limit irreversible disability.