COVID-19 Induces Neuroinflammation and Suppresses Peroxisomes in the Brain.

OBJECTIVE: Peroxisome injury occurs in the central nervous system (CNS) during multiple virus infections that result in neurological disabilities. We investigated host neuroimmune responses and peroxisome biogenesis factors during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using a multiplatform strategy. METHODS: Brain tissues from coronavirus disease 2019 (COVID-19) (n = 12) and other disease control (ODC) (n = 12) patients, as well as primary human neural cells and Syrian hamsters, infected with a clinical variant of SARS-CoV-2, were investigated by droplet digital polymerase chain reaction (ddPCR), quantitative reverse transcriptase PCR (RT-qPCR), and immunodetection methods. RESULTS: SARS-CoV-2 RNA was detected in the CNS of 4 patients with COVID-19 with viral protein (NSP3 and spike) immunodetection in the brainstem. Olfactory bulb, brainstem, and cerebrum from patients with COVID-19 showed induction of pro-inflammatory transcripts (IL8, IL18, CXCL10, NOD2) and cytokines (GM-CSF and IL-18) compared to CNS tissues from ODC patients (p < 0.05). Peroxisome biogenesis factor transcripts (PEX3, PEX5L, PEX11ß, and PEX14) and proteins (PEX3, PEX14, PMP70) were suppressed in the CNS of COVID-19 compared to ODC patients (p < 0.05). SARS-CoV-2 infection of hamsters revealed viral RNA detection in the olfactory bulb at days 4 and 7 post-infection while inflammatory gene expression was upregulated in the cerebrum of infected animals by day 14 post-infection (p < 0.05). Pex3 transcript levels together with catalase and PMP70 immunoreactivity were suppressed in the cerebrum of SARS-CoV-2 infected animals (p < 0.05). INTERPRETATION: COVID-19 induced sustained neuroinflammatory responses with peroxisome biogenesis factor suppression despite limited brainstem SARS-CoV-2 neurotropism in humans. These observations offer insights into developing biomarkers and therapies, while also implicating persistent peroxisome dysfunction as a contributor to the neurological post-acute sequelae of COVID-19. ANN NEUROL 2023;94:531-546.

Infection of Glia by Human Pegivirus Suppresses Peroxisomal and Antiviral Signaling Pathways.

Human pegivirus (HPgV) infects peripheral leukocytes but was recently shown to be a neurotropic virus associated with leukoencephalitis in humans. In the present study, we investigated the neural cell tropism of HPgV as well as its effects on host immune responses. HPgV wild type (WT) and a mutant virus with a deletion in the HPgV NS2 gene (ΔNS2) were able to productively infect human astrocytes and microglia but not neurons or an oligodendrocyte-derived cell line. Of note, the ΔNS2 virus replicated better than WT pegivirus in astrocytes, with both viruses being able to subsequently infect and spread in fresh human astrocyte cultures. Infection of human glia by HPgV WT and ΔNS2 viruses resulted in suppression of peroxisome-associated genes, including PEX11B, ABCD1, PEX7, ABCD3, PEX3, and PEX5L, during peak viral production, which was accompanied by reduced expression of IFNB, IRF3, IRF1, and MAVS, particularly in ΔNS2-infected cells. These data were consistent with analyses of brain tissue from patients infected with HPgV in which we observed suppression of peroxisome and type I interferon gene transcripts, including PEX11B, ABCD3, IRF1, and IRF3, with concurrent loss of PMP70 immunoreactivity in glia. Our data indicate that human astrocytes and microglia are permissive to HPgV infection, resulting in peroxisome injury and suppressed antiviral signaling that is influenced by viral diversity. IMPORTANCE Human pegiviruses are detected in 1 to 5% of the general population, principally infecting leukocytes, although their effects on human health remain uncertain. Here, we show that human pegivirus infects specific neural cell types in culture and human brain and, like other neurotropic flaviviruses, causes suppression of peroxisome and antiviral signaling pathways, which could favor ongoing viral infection and perhaps confer susceptibility to the development of neurological disease.

Absent in melanoma 2 regulates tumor cell proliferation in glioblastoma multiforme.

INTRODUCTION: Inflammation is a key aspect of glioblastoma multiforme (GBM) although it remains unclear how it contributes to GBM pathogenesis. Inflammasomes are intracellular multi-protein complexes that are involved in innate immunity and are activated by cellular stress, principally in macrophages. This study examined the expression of inflammasome-associated genes in GBM, particularly absent in melanoma 2 (AIM2). METHODS: Tissue samples from surgically-resected GBM tumors (n = 10) were compared to resected brain specimens from patients with epilepsy (age- and sex-matched Other Disease Controls (ODC, n=5)) by qRT-PCR, western blotting and immunofluorescence. Gene expression studies in human astrocytoma U251 cells were performed and the effects of deleting the absent in melanoma 2 (AIM2) gene using the CRISPR-Cas9 system were analyzed. RESULTS: GBM tissues showed significantly elevated expression of multiple immune (CD3E, CD163, CD68, MX1, ARG1) and inflammasome (AIM2, NLRP1, IL18, CASP1, and IL-33) genes compared to ODC tissues, without induction of IL1B, IFNG or TNFA. An insert-containing AIM2 variant transcript was highly expressed in GBM tissues and in U251 cells. AIM2 immunoreactivity was concentrated in the tumor core in the absence of PCNA immunodetection and showed a predominant 52 kDa immunoreactive band on western blot. Deletion of AIM2 resulted in significantly enhanced proliferation of U251 cells, which also displayed increased resistance to temozolomide treatment. CONCLUSIONS: GBM tumors express a distinct profile of inflammasome-associated genes in a tumor-specific manner. AIM2 expression in tumor cells suppressed cell proliferation while also conferring increased susceptibility to contemporary GBM therapy.

Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis.

Microbial communities reside in healthy tissues but are often disrupted during disease. Bacterial genomes and proteins are detected in brains from humans, nonhuman primates, rodents and other species in the absence of neurological disease. We investigated the composition and abundance of microbiota in frozen and fixed autopsied brain samples from patients with multiple sclerosis (MS) and age- and sex-matched nonMS patients as controls, using neuropathological, molecular and bioinformatics tools. 16s rRNA sequencing revealed Proteobacteria to be the dominant phylum with restricted diversity in cerebral white matter (WM) from MS compared to nonMS patients. Both clinical groups displayed 1,200-1,400 bacterial genomes/cm3 and low bacterial rRNA:rDNA ratios in WM. RNAseq analyses showed a predominance of Proteobacteria in progressive MS patients' WM, associated with increased inflammatory gene expression, relative to a broader range of bacterial phyla in relapsing-remitting MS patients' WM. Although bacterial peptidoglycan (PGN) and RNA polymerase beta subunit immunoreactivities were observed in all patients, PGN immunodetection was correlated with demyelination and neuroinflammation in MS brains. Principal component analysis revealed that demyelination, PGN and inflammatory gene expression accounted for 86% of the observed variance. Thus, inflammatory demyelination is linked to an organ-specific dysbiosis in MS that could contribute to underlying disease mechanisms.

GABA transport and neuroinflammation are coupled in multiple sclerosis: regulation of the GABA transporter-2 by ganaxolone.

Interactions between neurotransmitters and the immune system represent new prospects for understanding neuroinflammation and associated neurological disease. GABA is the chief inhibitory neurotransmitter but its actions on immune pathways in the brain are unclear. In the present study, we investigated GABAergic transport in conjunction with neuroinflammation in models of multiple sclerosis (MS). Protein and mRNA levels of γ-amino butyric acid transporter 2 (GAT-2) were examined in cerebral white matter from MS and control (Non-MS) patients, in cultured human macrophages, microglia and astrocytes, and in spinal cords from mice with and without experimental autoimmune encephalomyelitis (EAE) using western blotting, immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). GABA levels were measured by HPLC. The GAT-2's expression was increased in MS patients' (n=6) white matter, particularly in macrophage lineage cells, compared to Non-MS patients (n=6) (p<0.05). Interferon-γ (IFN-γ) stimulation of human macrophage lineage cells induced GAT-2 expression and reduced extracellular GABA levels (p<0.05) but soluble GABA treatment suppressed HLA-DRα, GAT-2 and XBP-1/s expression in stimulated macrophage lineage cells (p<0.05). Similarly, the synthetic allopregnanolone analog, ganaxolone (GNX), repressed GAT-2, JAK-1 and STAT-1 expression in activated macrophage lineage cells (p<0.05). In vivo GNX treatment reduced Gat-2, Cd3ε, MhcII, and Xbp-1/s expression in spinal cords following EAE induction (p<0.05), which was correlated with improved neurobehavioral outcomes and reduced neuroinflammation, demyelination and axonal injury. These findings highlight altered GABAergic transport through GAT-2 induction during neuroinflammation. GABA transport and neuroinflammation are closely coupled but regulated by GNX, pointing to GABAergic pathways as therapeutic targets in neuroinflammatory diseases.