Senescent-like microglia limit remyelination through the senescence associated secretory phenotype.

The capacity to regenerate myelin in the central nervous system (CNS) diminishes with age. This decline is particularly evident in multiple sclerosis (MS), which has been suggested to exhibit features of accelerated biological aging. Whether cellular senescence, a hallmark of aging, contributes to remyelination impairment remains unknown. Here, we show that senescent cells (SCs) accumulate within demyelinated lesions after injury, and their elimination enhances remyelination in young mice but not in aged mice. In young mice, we observed the upregulation of senescence-associated transcripts primarily in microglia after demyelination, followed by their reduction during remyelination. However, in aged mice, senescence-associated factors persisted within lesions, correlating with inefficient remyelination. We found that SC elimination enhanced remyelination in young mice but was ineffective in aged mice. Proteomic analysis of senescence-associated secretory phenotype (SASP) revealed elevated levels of CCL11/Eotaxin-1 in lesions, which was found to inhibit efficient oligodendrocyte maturation. These results suggest therapeutic targeting of SASP components, such as CCL11, may improve remyelination in aging and MS.

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

Micro-diffusely abnormal white matter: An early multiple sclerosis lesion phase with intensified myelin blistering.

OBJECTIVE: Multiple sclerosis (MS) is a chronic central nervous system disease whose white matter lesion origin remains debated. Recently, we reported subtle changes in the MS normal appearing white matter (NAWM), presenting with an increase in myelin blisters and myelin protein citrullination, which may recapitulate some of the prodromal degenerative processes involved in MS pathogenesis. Here, to clarify the relevance of these changes for subsequent MS myelin degeneration we explored their prevalence in WM regions characterized by subtly reduced myelination (dubbed as micro-diffusely abnormal white matter, mDAWM). METHODS: We used an in-depth (immuno)histochemistry approach in 27 MS donors with histological presence of mDAWM and 5 controls. An antibody panel against degenerative markers was combined and the presence of myelin/axonal aberrations was analyzed and compared with the NAWM from the same cases/slices/regions. RESULTS: mDAWM-defined areas exhibit ill-defined borders, no signs of Wallerian degeneration, and they associate with visible veins. Remarkably, such areas present with augmented myelin blister frequency, enhanced prevalence of polar myelin phospholipids, citrullination, and degradation of myelin basic protein (MBP) when compared with the NAWM. Furthermore, enhanced reactivity of microglia/macrophages against citrullinated MBP was also observed in this tissue. INTERPRETATION: We report a new histologically defined early phase in MS lesion formation, namely mDAWM, which lacks signs of Wallerian pathology. These results support the prelesional nature of the mDAWM. We conceptualize that evolution to pathologically evident lesions comprises the previously documented imbalance of axo-myelinic units (myelin blistering) leading to their degeneration and immune system activation by released myelin components.

Gasdermin D activation in oligodendrocytes and microglia drives inflammatory demyelination in progressive multiple sclerosis.

Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.

Microglia promote remyelination independent of their role in clearing myelin debris.

Multiple sclerosis (MS) is an inflammatory disease characterized by myelin loss. While therapies exist to slow MS progression, no treatment currently exists for remyelination. Remyelination, linked to reduced disability in MS, relies on microglia and monocyte-derived macrophages (MDMs). This study aims to understand the role of microglia during remyelination by lineage tracing and depleting them. Microglial lineage tracing reveals that both microglia and MDMs initially accumulate, but microglia later dominate the lesion. Microglia and MDMs engulf equal amounts of inhibitory myelin debris, but after microglial depletion, MDMs compensate by engulfing more myelin debris. Microglial depletion does, however, reduce the recruitment and proliferation of oligodendrocyte progenitor cells (OPCs) and impairs their subsequent differentiation and remyelination. These findings underscore the essential role of microglia during remyelination and offer insights for enhancing this process by understanding microglial regulation of remyelination.

Sex differences in peripheral immune cell activation: Implications for pain and pain resolution.

Decades of research into chronic pain has deepened our understanding of the cellular mechanisms behind this process. However, a failure to consider the biological variable of sex has limited the application of these breakthroughs into clinical application. In the present study, we investigate fundamental differences in chronic pain between male and female mice resulting from inflammatory activation of the innate immune system. We provide evidence that female mice are more sensitive to the effects of macrophages. Injecting small volumes of media conditioned by either unstimulated macrophages or macrophages stimulated by the inflammatory molecule TNFα lead to increased pain sensitivity only in females. Interestingly, we find that TNFα conditioned media leads to a more rapid resolution of mechanical hypersensitivity and altered immune cell recruitment to sites of injury. Furthermore, male and female macrophages exhibit differential polarization characteristics and motility after TNFα stimulation, as well as a different profile of cytokine secretions. Finally, we find that the X-linked gene Tlr7 is critical in the facilitating the adaptive resolution of pain in models of acute and chronic inflammation in both sexes. Altogether, these findings suggest that although the cellular mechanisms of pain resolution may differ between the sexes, the study of these differences may yield more targeted approaches with clinical applications.

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

Social isolation produces a sex- and brain region-specific alteration of microglia state.

Social isolation is a profound form of psychological stress that impacts the mental health of a large proportion of society. Other experimental models of stress have demonstrated a microglia response that serves either a protective or pathological function. However, the effect of adult social isolation on microglia has not been thoroughly investigated. We measured microglia territory, branching, end points and phagocytic-lysosomal activity in group housed C57Bl/6 mice and mice that were socially isolated for 2 weeks. Our results show that the dorsomedial hypothalamus and hippocampal CA2 region of adult male mice undergo increased microglia volume, territory and endpoints following social isolation, whereas females exhibit this increase in the hypothalamus only. Males exhibited decreases in the phagocytic-lysosomal marker CD68 in microglia in these regions, whereas females showed an increase in CD68 in the hypothalamus suggesting sexually dimorphic and brain region-specific change in microglia state in response to social isolation. The prefrontal cortex, central amygdala, nucleus accumbens shell and visual cortex did not exhibit changes in microglia structure in either male or female mice. These data show that microglia in different brain regions undergo a distinct response to social isolation which may account for changes in cognition and behaviour associated with this prevalent form of psychological stress.

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance.

BACKGROUND: Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. METHODS: To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. RESULTS: The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. CONCLUSIONS: Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

Sex differences in the inflammatory response of the mouse DRG and its connection to pain in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is an autoimmune disease with notable sex differences. Women are not only more likely to develop MS but are also more likely than men to experience neuropathic pain in the disease. It has been postulated that neuropathic pain in MS can originate in the peripheral nervous system at the level of the dorsal root ganglia (DRG), which houses primary pain sensing neurons (nociceptors). These nociceptors become hyperexcitable in response to inflammation, leading to peripheral sensitization and eventually central sensitization, which maintains pain long-term. The mouse model experimental autoimmune encephalomyelitis (EAE) is a good model for human MS as it replicates classic MS symptoms including pain. Using EAE mice as well as naïve primary mouse DRG neurons cultured in vitro, we sought to characterize sex differences, specifically in peripheral sensory neurons. We found sex differences in the inflammatory profile of the EAE DRG, and in the TNFα downstream signaling pathways activated intracellularly in cultured nociceptors. We also found increased cell death with TNFα treatment. Given that TNFα signaling has been shown to initiate intrinsic apoptosis through mitochondrial disruption, this led us to investigate sex differences in the mitochondria's response to TNFα. Our results demonstrate that male sensory neurons are more sensitive to mitochondrial stress, making them prone to neuronal injury. In contrast, female sensory neurons appear to be more resistant to mitochondrial stress and exhibit an inflammatory and regenerative phenotype that may underlie greater nociceptor hyperexcitability and pain. Understanding these sex differences at the level of the primary sensory neuron is an important first step in our eventual goal of developing sex-specific treatments to halt pain development in the periphery before central sensitization is established.

Towards a definition of microglia heterogeneity.

High dimensional single-cell analysis such as single cell and single nucleus RNA sequencing (sc/snRNAseq) are currently being widely applied to explore microglia diversity. The use of sc/snRNAseq provides a powerful and unbiased approach to deconvolve heterogeneous cellular populations. However, sc/snRNAseq and analyses pipelines are designed to find heterogeneity. Indeed, cellular heterogeneity is often the most frequently reported finding. In this Perspective, we consider the ubiquitous concept of heterogeneity focusing on its application to microglia research and its influence on the field of neuroimmunology. We suggest that a clear understanding of the semantic and biological implications of microglia heterogeneity is essential for mitigating confusion among researchers.

Oligodendrocyte death and myelin loss in the cuprizone model: an updated overview of the intrinsic and extrinsic causes of cuprizone demyelination.

BACKGROUND: The dietary consumption of cuprizone - a copper chelator - has long been known to induce demyelination of specific brain structures and is widely used as model of multiple sclerosis. Despite the extensive use of cuprizone, the mechanism by which it induces demyelination are still unknown. With this review we provide an updated understanding of this model, by showcasing two distinct yet overlapping modes of action for cuprizone-induced demyelination; 1) damage originating from within the oligodendrocyte, caused by mitochondrial dysfunction or reduced myelin protein synthesis. We term this mode of action 'intrinsic cell damage'. And 2) damage to the oligodendrocyte exerted by inflammatory molecules, brain resident cells, such as oligodendrocytes, astrocytes, and microglia or peripheral immune cells - neutrophils or T-cells. We term this mode of action 'extrinsic cellular damage'. Lastly, we summarize recent developments in research on different forms of cell death induced by cuprizone, which could add valuable insights into the mechanisms of cuprizone toxicity. With this review we hope to provide a modern understanding of cuprizone-induced demyelination to understand the causes behind the demyelination in MS.

Central nervous system macrophages in progressive multiple sclerosis: relationship to neurodegeneration and therapeutics.

There are over 15 disease-modifying drugs that have been approved over the last 20 years for the treatment of relapsing-remitting multiple sclerosis (MS), but there are limited treatment options available for progressive MS. The development of new drugs for the treatment of progressive MS remains challenging as the pathophysiology of progressive MS is poorly understood.The progressive phase of MS is dominated by neurodegeneration and a heightened innate immune response with trapped immune cells behind a closed blood-brain barrier in the central nervous system. Here we review microglia and border-associated macrophages, which include perivascular, meningeal, and choroid plexus macrophages, during the progressive phase of MS. These cells are vital and are largely the basis to define lesion types in MS. We will review the evidence that reactive microglia and macrophages upregulate pro-inflammatory genes and downregulate homeostatic genes, that may promote neurodegeneration in progressive MS. We will also review the factors that regulate microglia and macrophage function during progressive MS, as well as potential toxic functions of these cells. Disease-modifying drugs that solely target microglia and macrophage in progressive MS are lacking. The recent treatment successes for progressive MS include include B-cell depletion therapies and sphingosine-1-phosphate receptor modulators. We will describe several therapies being evaluated as a potential treatment option for progressive MS, such as immunomodulatory therapies that can target myeloid cells or as a potential neuroprotective agent.

Myelin Quantification in White Matter Pathology of Progressive Multiple Sclerosis Post-Mortem Brain Samples: A New Approach for Quantifying Remyelination.

Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the central nervous system (CNS). Repair through remyelination can be extensive, but quantification of remyelination remains challenging. To date, no method for standardized digital quantification of remyelination of MS lesions exists. This methodological study aims to present and validate a novel standardized method for myelin quantification in progressive MS brains to study myelin content more precisely. Fifty-five MS lesions in 32 tissue blocks from 14 progressive MS cases and five tissue blocks from 5 non-neurological controls were sampled. MS lesions were selected by macroscopic investigation of WM by standard histopathological methods. Tissue sections were stained for myelin with luxol fast blue (LFB) and histological assessment of de- or remyelination was performed by light microscopy. The myelin quantity was estimated with a novel myelin quantification method (MQM) in ImageJ. Three independent raters applied the MQM and the inter-rater reliability was calculated. We extended the method to diffusely appearing white matter (DAWM) and encephalitis to test potential wider applicability of the method. Inter-rater agreement was excellent (ICC = 0.96) and there was a high reliability with a lower- and upper limit of agreement up to -5.93% to 18.43% variation in myelin quantity. This study builds on the established concepts of histopathological semi-quantitative assessment of myelin and adds a novel, reliable and accurate quantitative measurement tool for the assessment of myelination in human post-mortem samples.

An X-ray for myelin.

With substantial progress in experimental therapeutics to enhance the nervous system's capacity for remyelination, new methods to detect myelin are important advances. We discuss a small-angle X-ray scattering tensor tomography approach presented recently by Georgiadis et al. and the method's promise in providing a new window into the brain to evaluate myelin integrity in health and disease.

Regulation of microglia population dynamics throughout development, health, and disease.

The dynamic expansions and contractions of the microglia population in the central nervous system (CNS) to achieve homeostasis are likely vital for their function. Microglia respond to injury or disease but also help guide neurodevelopment, modulate neural circuitry throughout life, and direct regeneration. Throughout these processes, microglia density changes, as does the volume of area that each microglia surveys. Given that microglia are responsible for sensing subtle alterations to their environment, a change in their density could affect their capacity to mobilize rapidly. In this review, we attempt to synthesize the current literature on the ligands and conditions that promote microglial proliferation across development, adulthood, and neurodegenerative conditions. Microglia display an impressive proliferative capacity during development and in neurodegenerative diseases that is almost completely absent at homeostasis. However, the appropriate function of microglia in each state is critically dependent on density fluctuations that are primarily induced by proliferation. Proliferation is a natural microglial response to insult and often serves neuroprotective functions. In contrast, inappropriate microglial proliferation, whether too much or too little, often precipitates undesirable consequences for nervous system health. Thus, fluctuations in the microglia population are tightly regulated to ensure these immune cells can execute their diverse functions.

The CD33 short isoform is a gain-of-function variant that enhances Aß1-42 phagocytosis in microglia.

BACKGROUND: CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant. METHODS: We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently. RESULTS: In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding. CONCLUSIONS: These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.