Brain macrophages acquire distinct transcriptomes in multiple sclerosis lesions and normal appearing white matter.

Multiple sclerosis (MS) is a disease of the central nervous system that is characterized by inflammation and focal areas of demyelination, ultimately resulting in axonal degradation and neuronal loss. Several lines of evidence point towards a role for microglia and other brain macrophages in disease initiation and progression, but exactly how lesion formation is triggered is currently unknown. Here, we characterized early changes in MS brain tissue through transcriptomic analysis of normal appearing white matter (NAWM). We found that NAWM was characterized by enriched expression of genes associated with inflammation and cellular stress derived from brain macrophages. Single cell RNA sequencing confirmed a stress response in brain macrophages in NAWM and identified specific microglia and macrophage subsets at different stages of demyelinating lesions. We identified both phagocytic/activated microglia and CAM clusters that were associated with various MS lesion types. These overall changes in microglia and macrophages associated with lesion development in MS brain tissue may provide therapeutic targets to limit lesion progression and demyelination.

The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer's disease.

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer's disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC-MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy.

Ageing and recurrent episodes of neuroinflammation promote progressive experimental autoimmune encephalomyelitis in Biozzi ABH mice.

Current therapies for multiple sclerosis (MS) reduce the frequency of relapses by modulating adaptive immune responses but fail to limit the irreversible neurodegeneration driving progressive disability. Experimental autoimmune encephalomyelitis (EAE) in Biozzi ABH mice recapitulates clinical features of MS including relapsing-remitting episodes and secondary-progressive disability. To address the contribution of recurrent inflammatory events and ageing as factors that amplify progressive neurological disease, we examined EAE in 8- to 12-week-old and 12-month-old ABH mice. Compared with the relapsing-remitting (RREAE) and secondary progressive (SPEAE) EAE observed in young mice, old mice developed progressive disease from onset (PEAE) associated with pronounced axonal damage and increased numbers of CD3(+) T cells and microglia/macrophages, but not B cells. Whereas the clinical neurological features of PEAE and SPEAE were comparable, the pathology was distinct. SPEAE was associated with significantly reduced perivascular infiltrates and T-cell numbers in the central nervous system (CNS) compared with PEAE and the acute phase of RREAE. In contrast to perivascular infiltrates that declined during progression from RREAE into SPEAE, the numbers of microglia clusters remained constant. Similar to what is observed during MS, the microglia clusters emerging during EAE were associated with axonal damage and oligodendrocytes expressing heat-shock protein B5, but not lymphocytes. Taken together, our data reveal that the course of EAE is dependent on the age of the mice. Younger mice show a relapsing-remitting phase followed by progressive disease, whereas old mice immediately show progression. This indicates that recurrent episodes of inflammation in the CNS, as well as age, contribute to progressive neurological disease.

Pentraxin-3 is upregulated in the central nervous system during MS and EAE, but does not modulate experimental neurological disease.

Pentraxin-3 (PTX3), an acute-phase protein released during inflammation, aids phagocytic clearance of pathogens and apoptotic cells, and plays diverse immunoregulatory roles in tissue injury. In neuroinflammatory diseases, like MS, resident microglia could become activated by endogenous agonists for Toll like receptors (TLRs). Previously we showed a strong TLR2-mediated induction of PTX3 in cultured human microglia and macrophages by HspB5, which accumulates in glia during MS. Given the anti-inflammatory effects of HspB5, we examined the contribution of PTX3 to these effects in MS and its animal model EAE. Our data indicate that TLR engagement effectively induces PTX3 expression in human microglia, and that such expression is readily detectable in MS lesions. Enhanced PTX3 expression is prominently expressed in microglia in preactive MS lesions, and in microglia/macrophages engaged in myelin phagocytosis in actively demyelinating lesions. Yet, we did not detect PTX3 in cerebrospinal fluid of MS patients. PTX3 expression is also elevated in spinal cords during chronic relapsing EAE in Biozzi ABH mice, but the EAE severity and time course in PTX3-deficient mice did not differ from WT mice. Moreover, systemic PTX3 administration did not alter the disease onset or severity. Our findings reveal local functions of PTX3 during neuroinflammation in facilitating myelin phagocytosis, but do not point to a role for PTX3 in controlling the development of autoimmune neuroinflammation.

Small heat shock proteins are induced during multiple sclerosis lesion development in white but not grey matter.

INTRODUCTION: The important protective role of small heat-shock proteins (HSPs) in regulating cellular survival and migration, counteracting protein aggregation, preventing apoptosis, and regulating inflammation in the central nervous system is now well-recognized. Yet, their role in the neuroinflammatory disorder multiple sclerosis (MS) is largely undocumented. With the exception of alpha B-crystallin (HSPB5), little is known about the roles of small HSPs in disease. RESULTS: Here, we examined the expression of four small HSPs during lesion development in MS, focussing on their cellular distribution, and regional differences between white matter (WM) and grey matter (GM). It is well known that MS lesions in these areas differ markedly in their pathology, with substantially more intense blood-brain barrier damage, leukocyte infiltration and microglial activation typifying WM but not GM lesions. We analysed transcript levels and protein distribution profiles for HSPB1, HSPB6, HSPB8 and HSPB11 in MS lesions at different stages, comparing them with normal-appearing brain tissue from MS patients and non-neurological controls. During active stages of demyelination in WM, and especially the centre of chronic active MS lesions, we found significantly increased expression of HSPB1, HSPB6 and HSPB8, but not HSPB11. When induced, small HSPs were exclusively found in astrocytes but not in oligodendrocytes, microglia or neurons. Surprisingly, while the numbers of astrocytes displaying high expression of small HSPs were markedly increased in actively demyelinating lesions in WM, no such induction was observed in GM lesions. This difference was particularly obvious in leukocortical lesions covering both WM and GM areas. CONCLUSIONS: Since induction of small HSPs in astrocytes is apparently a secondary response to damage, their differential expression between WM and GM likely reflects differences in mediators that accompany demyelination in either WM or GM during MS. Our findings also suggest that during MS, cortical structures fail to benefit from the protective actions of small HSPs.

Activation status of human microglia is dependent on lesion formation stage and remyelination in multiple sclerosis.

Similar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis-affected CNS tissues and underscore the plasticity of human adult microglia in vitro.

Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis.

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

Characterization of immune response to neurofilament light in experimental autoimmune encephalomyelitis.

BACKGROUND: Autoimmunity to neuronal proteins occurs in several neurological syndromes, where cellular and humoral responses are directed to surface as well as intracellular antigens. Similar to myelin autoimmunity, pathogenic immune response to neuroaxonal components such as neurofilaments may contribute to neurodegeneration in multiple sclerosis. METHODS: We studied the immune response to the axonal protein neurofilament light (NF-L) in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. To examine the association between T cells and axonal damage, pathology studies were performed on NF-L immunized mice. The interaction of T cells and axons was analyzed by confocal microscopy of central nervous system tissues and T-cell and antibody responses to immunodominant epitopes identified in ABH (H2-Ag7) and SJL/J (H2-As) mice. These epitopes, algorithm-predicted peptides and encephalitogenic motifs within NF-L were screened for encephalitogenicity. RESULTS: Confocal microscopy revealed both CD4+ and CD8+ T cells alongside damaged axons in the lesions of NF-L immunized mice. CD4+ T cells dominated the areas of axonal injury in the dorsal column of spastic mice in which the expression of granzyme B and perforin was detected. Identified NF-L epitopes induced mild neurological signs similar to the observed with the NF-L protein, yet distinct from those characteristic of neurological disease induced with myelin oligodendrocyte glycoprotein. CONCLUSIONS: Our data suggest that CD4+ T cells are associated with spasticity, axonal damage and neurodegeneration in NF-L immunized mice. In addition, defined T-cell epitopes in the NF-L protein might be involved in the pathogenesis of the disease.

Clusters of activated microglia in normal-appearing white matter show signs of innate immune activation.

BACKGROUND: In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples. METHODS: Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions. RESULTS: Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters. CONCLUSIONS: The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.