Pericytes as mediators of infiltration of macrophages in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative condition of the central nervous system (CNS). It is associated with blood-brain barrier (BBB) breakdown and intravasation of leukocytes, particularly monocyte-derived macrophages, into the CNS. Pericytes are mural cells that are encased within the basement membrane of vasculature, and they contribute functionally to the neurovascular unit. These cells play an important role in maintaining BBB integrity and CNS homeostasis. However, the critical role of pericytes in mediating inflammation in MS or its models is unclear. Whether pericytes infiltrate into the CNS parenchyma in MS also needs clarification. METHODS: CNS samples from the experimental autoimmune encephalomyelitis (EAE) mouse model of MS were collected at different time points for immunohistochemical analysis of pericytes along the inflamed vasculature. These findings were validated using MS brain specimens, and further analysis of pericyte involvement in inflammation was carried out by culturing primary pericytes and macrophages. Multiplex ELISA, transmigration assay and real-time PCR were used to study the inflammatory potential of pericytes in cultures. RESULTS: We found that pericytes exhibit a heterogenous morphology, with notable elongation in the inflamed perivascular cuffs of EAE. This was manifested by a decrease in pericyte density but an increase in the coverage by pericytes along the vasculature. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix proteins enriched within inflamed perivascular cuffs, elevated levels of pro-inflammatory chemokines/cytokines in pericytes in culture. Importantly, pericytes stimulated with CSPGs enhanced macrophage migration. We did not detect pericytes in the CNS parenchyma during EAE, and this was corroborated in MS brain samples. CONCLUSIONS: Our data suggest that pericytes seek to restore the BBB through increased coverage, but that their exposure to CSPGs prompt their facilitation of macrophages to enter the CNS to elevate neuroinflammation in EAE and MS.

Biochemically altered myelin triggers autoimmune demyelination.

Although immune attack against central nervous system (CNS) myelin is a central feature of multiple sclerosis (MS), its root cause is unresolved. In this report, we provide direct evidence that subtle biochemical modifications to brain myelin elicit pathological immune responses with radiological and histological properties similar to MS lesions. A subtle myelinopathy induced by abbreviated cuprizone treatment, coupled with subsequent immune stimulation, resulted in lesions of inflammatory demyelination. The degree of myelin injury dictated the resulting immune response; biochemical damage that was too limited or too extensive failed to trigger overt pathology. An inhibitor of peptidyl arginine deiminases (PADs), enzymes that alter myelin structure and correlate with MS lesion severity, mitigated pathology even when administered only during the myelin-altering phase. Moreover, cultured splenocytes were reactive against donor myelin isolates, a response that was substantially muted when splenocytes were exposed to myelin from donors treated with PAD inhibitors. By showing that a primary biochemical myelinopathy can trigger secondary pathological inflammation, "cuprizone autoimmune encephalitis" potentially reconciles conflicting theories about MS pathogenesis and provides a strong rationale for investigating myelin as a primary target for early, preventative therapy.

The intestinal barrier in multiple sclerosis: implications for pathophysiology and therapeutics.

Biological barriers are essential for the maintenance of homeostasis in health and disease. Breakdown of the intestinal barrier is an essential aspect of the pathophysiology of gastrointestinal inflammatory diseases, such as inflammatory bowel disease. A wealth of recent studies has shown that the intestinal microbiome, part of the brain-gut axis, could play a role in the pathophysiology of multiple sclerosis. However, an essential component of this axis, the intestinal barrier, has received much less attention. In this review, we describe the intestinal barrier as the physical and functional zone of interaction between the luminal microbiome and the host. Besides its essential role in the regulation of homeostatic processes, the intestinal barrier contains the gut mucosal immune system, a guardian of the integrity of the intestinal tract and the whole organism. Gastrointestinal disorders with intestinal barrier breakdown show evidence of CNS demyelination, and content of the intestinal microbiome entering into the circulation can impact the functions of CNS microglia. We highlight currently available studies suggesting that there is intestinal barrier dysfunction in multiple sclerosis. Finally, we address the mechanisms by which commonly used disease-modifying drugs in multiple sclerosis could alter the intestinal barrier and the microbiome, and we discuss the potential of barrier-stabilizing strategies, including probiotics and stabilization of tight junctions, as novel therapeutic avenues in multiple sclerosis.

Genetically predicted hypotaurine levels mediate the relationship between immune cells and intracerebral hemorrhage.

The evidence supports a strong link between immune cells and intracerebral hemorrhage (ICH). Nonetheless, the specific cause-and-effect associations between immune cells and ICH remain indeterminate. Here, our primary investigation compared immune cell infiltration in the ICH and sham groups using the GSE24265 dataset. Afterward, we extensively examined the relationship between immune cells and ICH by applying a two-sample Mendelian randomization (MR) analysis to identify the particular immune cells that may be associated with the initiation and advancement of ICH. Nevertheless, the specific processes that regulate the cause-and-effect connection between immune cells and ICH remain unknown. In this study, our objective was to investigate the connections between immune cell characteristics and plasma metabolites, as well as the links between plasma components and ICH. Our investigation uncovered that the levels of hypotaurine play a key role in the advancement of ICH, influencing the ratio of switched memory B cells among lymphocytes. Thus, our findings provide novel insights into the potential biological mechanisms underlying immune cell-mediated ICH.

Vildagliptin improves neurological function by inhibiting apoptosis and ferroptosis following intracerebral hemorrhage in mice.

Intracerebral hemorrhage (ICH) is a fatal health problem which lacks effective treatment. The apoptosis caused by hematoma constituents, and the ferroptosis due to iron overload, are prominent contributors of neurologic impairment after ICH. Targeting cell death pathways may thus be a therapeutic strategy for neuroprotection and functional recovery in ICH. Vildagliptin (Vilda), a dipeptidyl peptidase (DPP)-4 inhibitor, has been reported to have potent anti-apoptosis and anti-ferroptotic capacity. However, it is not clear whether Vilda has anti-cell death efficacy in ICH. In the present study, the potential neuroprotective effect of Vilda in ICH mice was investigated. Mice were randomly divided into three groups: sham, ICH + saline or ICH + Vilda. ICH was induced by collagenase type VII micro-injection into the right basal ganglia. Vilda (50 mg/kg/day; gavage) daily treatment for 3 days after ICH improved neurological deficit scores, reduced hematoma volume, and inhibited degeneration of neurons. The activation of microglia/macrophages and infiltration of neutrophil were restrained by Vilda. Moreover, Vilda attenuated brain cell apoptosis as determined by TUNEL staining, raised Bcl-2 protein level, and simultaneously suppressed Bax as validated by western blots. In addition, Vilda reduced malondialdehyde level, elevated glutathione peroxidase brain content, and alleviated iron deposition at 3 days after ICH in mice. In conclusion, Vilda exerts neuroprotective effects in ICH, at least in part by inhibiting neuroinflammation, and preventing neuronal apoptosis and ferroptosis following ICH.

Expression of antioxidant enzymes in lesions of multiple sclerosis and its models.

Oxidative stress promotes tissue injury in the central nervous system in neurological disorders such as multiple sclerosis (MS). To protect against this, antioxidant enzymes including superoxide dismutase-1 (SOD1), heme oxygenase-1 (HO-1), peroxiredoxin-5 (PRDX5) and glutathione peroxidase-4 (GPX4) may be upregulated. However, whether antioxidant enzyme elevation in mouse models of neurodegeneration corresponds to their expression in human diseases such as MS requires investigation. Here, we analyzed and compared the expression of SOD1, HO-1, PRDX5 and GPX4 in the murine spinal cord of three models of MS: focal lesions induced by (1) oxidized phosphatidylcholine or (2) lysophosphatidylcholine (lysolecithin), and (3) diffuse lesions of experimental autoimmune encephalomyelitis. Notably, CD68+ microglia/macrophages were the predominant cellular populations that expressed the highest levels of the detected antioxidant enzymes. Overall, the expression patterns of antioxidant enzymes across the models were similar. The increase of these antioxidant enzymes was corroborated in MS brain tissue using spatial RNA sequencing. Collectively, these results show that antioxidant capacity is relatively conserved between mouse models and MS lesions, and suggest a need to investigate whether the antioxidant elevation in microglia/macrophages is a protective response during oxidative injury, neurodegeneration, and MS.

Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.

Chronic mild stress exacerbates severity of experimental autoimmune encephalomyelitis in association with altered non-coding RNA and metabolic biomarkers.

The causal factors determining the onset and severity of multiple sclerosis (MS) are not well understood. Here, we investigated the influence of chronic stress on clinical symptoms, metabolic and epigenetic manifestations of experimental autoimmune encephalomyelitis (EAE), a common animal model of MS. Lewis rats were immunized for monophasic EAE with MBP69-88 and were exposed to chronic stress for 37days starting 7days prior to immunization. The exposure to stress accelerated and exacerbated the clinical symptoms of EAE. Both stress and EAE also disrupted metabolic status as indicated by trace elemental analysis in body hair. Stress particularly exacerbated chlorine deposition in EAE animals. Moreover, deep sequencing revealed a considerable impact of stress on microRNA expression in EAE. EAE by itself upregulated microRNA expression in lumbar spinal cord, including miR-21, miR-142-3p, miR-142-5p, miR-146a, and miR-155. Stress in EAE further up-regulated miR-16, miR-146a and miR-155 levels. The latter two microRNAs are recognized biomarkers of human MS. Thus, stress may synergistically exacerbate severity of EAE by altering epigenetic regulatory pathways. The findings suggest that stress may represent a significant risk factor for symptomatic deterioration in MS. Stress-related metabolic and microRNA signatures support their value as biomarkers for predicting the risk and severity of MS.

Multi-target-directed phenol-triazole ligands as therapeutic agents for Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disease that is characterized by the formation of intracellular neurofibrillary tangles and extracellular amyloid-ß (Aß) plaque deposits. Increased oxidative stress, metal ion dysregulation, and the formation of toxic Aß peptide oligomers are all considered to contribute to the etiology of AD. In this work we have developed a series of ligands that are multi-target-directed in order to address several disease properties. 2-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)phenol (POH), 2-(1-(2-morpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PMorph), and 2-(1-(2-thiomorpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PTMorph) have been synthesized and screened for their antioxidant capacity, Cu-binding affinity, interaction with the Aß peptide and modulation of Aß peptide aggregation, and the ability to limit Aß1-42-induced neurotoxicity in human neuronal culture. The synthetic protocol and structural variance incorporated via click chemistry, highlights the influence of R-group modification on ligand-Aß interactions and neuroprotective effects. Overall, this study demonstrates that the phenol-triazole ligand scaffold can target multiple factors associated with AD, thus warranting further therapeutic development.

Gray Matter Hypoxia in the Brain of the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) has a significant inflammatory component and may have significant gray matter (GM) pathophysiology. Brain oxygenation is a sensitive measurement of the balance between metabolic need and oxygen delivery. There is evidence that inflammation and hypoxia are interdependent. In this paper, we applied novel, implanted PO2 sensors to measure hypoxia in cortical and cerebellar GM, in an inflammation-induced mouse model of MS. OBJECTIVE: Quantify oxygenation in cortical and cerebellar GM in the awake, unrestrained experimental autoimmune encephalomyelitis (EAE) mouse model and to relate the results to symptom level and disease time-course. METHODS: C57BL/6 mice were implanted with a fiber-optic sensor in the cerebellum (n = 13) and cortex (n = 24). Animals were induced with stimulation of the immune response and sensitization to myelin oligodendrocyte glycoprotein (MOG). Controls did not have MOG. We measured PO2 in awake, unrestrained animals from pre-induction (baseline) up to 36 days post-induction for EAE and controls. RESULTS: There were more days with hypoxia than hyperoxia (cerebellum: 34/67 vs. 18/67 days; cortex: 85/112 vs. 22/112) compared to time-matched controls. The average decline in PO2 on days that were significantly lower than time-matched controls was -8.8±6.0 mmHg (mean ± SD) for the cerebellum and -8.0±4.6 for the cortex. Conversely, the average increase in PO2 on days that were significantly hyperoxic was +3.2±2.8 mmHg (mean ± SD) for the cerebellum and +0.8±2.1 for the cortex. Cortical hypoxia related to increased behavioral deficits. Evidence for hypoxia occurred before measurable behavioral deficits. CONCLUSIONS: A highly inflammatory condition primed to a white matter (WM) autoimmune response correlates with significant hypoxia and increased variation in oxygenation in GM of both cerebellum and cortex in the mouse EAE model of MS.

Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis.

BACKGROUND: Microglial activation is thought to be a key pathophysiological mechanism underlying disease activity in all forms of MS. Hydroxychloroquine (HCQ) is an antimalarial drug with immunomodulatory properties that is widely used in the treatment of rheumatological diseases. In this series of experiments, we explore the effect of HCQ on human microglial activation in vitro and on the development of experimental autoimmune encephalitis (EAE) in vivo. METHODS: We activated human microglia with lipopolysaccharide (LPS), and measured concentrations of several pro- and anti-inflammatory cytokines in untreated and HCQ pretreated cultures. We investigated the effect of HCQ pretreatment at two doses on the development of EAE and spinal cord histology. RESULTS: HCQ pretreatment reduced the production of pro-inflammatory (TNF-alpha, IL-6, and IL-12) and anti-inflammatory (IL-10 and IL-1 receptor antagonist) cytokines in LPS-stimulated human microglia. HCQ pretreatment delayed the onset of EAE, and reduced the number of Iba-1 positive microglia/macrophages and signs of demyelination in the spinal cords of HCQ treated animals. CONCLUSION: HCQ treatment reduces the activation of human microglia in vitro, delays the onset of EAE, and decreases the representation of activated macrophages/microglia and demyelination in the spinal cord of treated mice. HCQ is a plausible candidate for further clinical studies in MS.

Serum NSE level and disability progression in multiple sclerosis.

BACKGROUND: Previous studies suggested that serum neuron specific enolase (NSE) may be a biomarker associated with progression in MS. METHODS: We measured serum NSE levels in 385 patients with multiple sclerosis (MS) (264 with relapsing-remitting (RR) MS, 86 with secondary progressive (SP) MS, and 35 with primary progressive (PP) MS), and compared levels between disease courses, between users and non-users of immunomodulatory treatment, and between patients with worsening or stable disability at one year follow-up (available in 161 patients). We also investigated the correlation between serum NSE and Expanded Disability Status Scale (EDSS) and MS Severity Score (MSSS) scores in the whole cohort and in subgroups, and built a multiple linear regression model to assess the influence of predictor variables on serum NSE. RESULTS: Age was the only independent predictor of serum NSE levels in the multiple linear regression model. In the subgroup of patients with PPMS, there was a moderate correlation between serum NSE and increasing MSSS (Pearson's r 0.35, p=0.04) and EDSS (Spearman's rho 0.37, p=0.03) scores. CONCLUSION: Our data do not support the use of serum NSE as a prognostic biomarker in RRMS or SPMS. The correlations of serum NSE with EDSS and MSSS in the PPMS subgroup are interesting, but based on a small sample size and require replication in other cohorts.

Immune modulatory therapies for spinal cord injury--past, present and future.

Historically, the immune response after spinal cord injury was considered largely detrimental owing to the release of neurotoxic factors. While there is validity to this view, there is much greater heterogeneity of immune cells than was previously realized. Associated with this heterogeneity of immune cell subtypes, there is diversity of functions of immune cells that is still poorly understood after spinal cord injury. Modulating the immune system requires improved understanding of the major players: those immune cell subtypes that are more detrimental than beneficial and those that are important in repair. In this review we will discuss the early findings that supported the use of various anti-inflammatory medications as well as the evolving concept that not all immune subtypes are detrimental and some might even be beneficial. In the last section we will highlight the need to characterize better the role of immune cell subsets in the hopes of developing potential therapeutic targets for the future.

Harmful and beneficial effects of inflammation after spinal cord injury: potential therapeutic implications.

Spinal cord injury (SCI) results in immediate damage followed by a secondary phase of tissue damage that occurs over a period of several weeks. The mechanisms underlying this secondary damage are multiple and not fully understood. A number of studies suggest that the local inflammatory response in the spinal cord that occurs after SCI contributes importantly to secondary damage. This response is mediated by cells normally found in the central nervous system (CNS) as well as infiltrating leukocytes. While the inflammatory response mediated by these cells is required for efficient clearance of tissue debris, and promotes wound healing and tissue repair, they also release various factors that can be detrimental to neurons, glia, axons, and myelin. In this chapter we provide an overview of the inflammatory response at the cell and molecular level after SCI, and review the current state of knowledge about its contribution to tissue damage and repair. Additionally, we discuss how some of this work is leading to the development and testing of drugs that modulate inflammation to treat acute SCI in humans.

Inflammation in neurological disorders: a help or a hindrance?

Inflammation of the central nervous system (CNS) (neuroinflammation) is now recognized to be a feature of all neurological disorders. In multiple sclerosis, there is prominent infiltration of various leukocyte subsets into the CNS. Even when there is no significant inflammatory infiltrates, such as in Parkinson or Alzheimer disease, there is intense activation of microglia with resultant elevation of many inflammatory mediators within the CNS. An extensive dataset describes neuroinflammation to have detrimental consequences, but results emerging largely over the past decade have indicated that aspects of the inflammatory response are beneficial for CNS outcomes. Benefits of neuroinflammation now include neuroprotection, the mobilization of neural precursors for repair, remyelination, and even axonal regeneration. The findings that neuroinflammation can be beneficial should not be surprising as a properly directed inflammatory response in other tissues is a natural healing process after an insult. In this article, we review the data that highlight the dual aspects of neuroinflammation in being a hindrance on the one hand but also a significant help for recovery of the CNS on the other. We consider how the inflammatory response may be beneficial or injurious, and we describe strategies to harness the beneficial aspects of neuroinflammation.