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Chronic exposure to manganese (Mn) is associated with neuroinflammation and extrapyramidal motor deficits resembling features of Parkinson’s disease. Activation of astrocytes and microglia is implicated in neuronal injury from Mn but it is not known whether early life exposure to Mn may predispose glia to more severe inflammatory responses during aging. We therefore examined astrocyte nuclear factor kappa B (NF-κB) signaling in mediating innate immune inflammatory responses during multiple neurotoxic exposures spanning juvenile development into adulthood. MnCl2 was given in drinking water for 30-day postweaning to both wildtype mice and astrocyte-specific knockout (KO) mice lacking I kappa B kinase 2, the central upstream activator of NF-κB. Following juvenile exposure to Mn, mice were subsequently administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 4 months of age. Animals were evaluated for behavioral alterations and brain tissue was analyzed for catecholamine neurotransmitters. Stereological analysis of neuronal and glial cell counts from multiple brain regions indicated that juvenile exposure to Mn amplified glial activation and neuronal loss from MPTP exposure in the caudate-putamen and globus pallidus, as well as increased the severity of neurobehavioral deficits in open field activity assays. These alterations were prevented in astrocyte-specific I kappa B kinase 2 KO mice. Juvenile exposure to Mn increased the number of neurotoxic A1 astrocytes expressing C3 as well as the number of activated microglia in adult mice following MPTP challenge, both of which were inhibited in KO mice. These results demonstrate that exposure to Mn during juvenile development heightens the innate immune inflammatory response in glia during a subsequent neurotoxic challenge through NF-κB signaling in astrocytes.

Infection with Influenza A virus can lead to the development of encephalitis and subsequent neurological deficits ranging from headaches to neurodegeneration. Post-encephalitic parkinsonism has been reported in surviving patients of H1N1 infections, but not all cases of encephalitic H1N1 infection present with these neurological symptoms, suggesting that interactions with an environmental neurotoxin could promote more severe neurological damage. The heavy metal, manganese (Mn), is a potential interacting factor with H1N1 because excessive exposure early in life can induce long-lasting effects on neurological function through inflammatory activation of glial cells. In the current study, we used a two-hit model of neurotoxin-pathogen exposure to examine whether exposure to Mn during juvenile development would induce a more severe neuropathological response following infection with H1N1 in adulthood. To test this hypothesis, C57BL/6 mice were exposed to MnCl2 in drinking water (50 mg/kg/day) for 30 days from days 21–51 postnatal, then infected intranasally with H1N1 three weeks later. Analyses of dopaminergic neurons, microglia and astrocytes in basal ganglia indicated that although there was no significant loss of dopaminergic neurons within the substantia nigra pars compacta, there was more pronounced activation of microglia and astrocytes in animals sequentially exposed to Mn and H1N1, as well as altered patterns of histone acetylation. Whole transcriptome Next Generation Sequencing (RNASeq) analysis was performed on the substantia nigra and revealed unique patterns of gene expression in the dual-exposed group, including genes involved in antioxidant activation, mitophagy and neurodegeneration. Taken together, these results suggest that exposure to elevated levels of Mn during juvenile development could sensitize glial cells to more severe neuro-immune responses to influenza infection later in life through persistent epigenetic changes.

Significance Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease of aging, affecting approximately 10 million patients worldwide with no approved therapies to modify progression of disease. Further understanding of the cellular mechanisms contributing to the development of PD is necessary to discover therapies. Here, we characterize the role of a recently identified GWAS hit for sporadic PD, ITPKB, in the aggregation of α-synuclein, the primary pathological feature of disease. These results identify inhibition of inositol-1,4,5,-triphosphate (IP3)-mediated ER-to-mitochondria calcium release as a potential therapeutic approach for reducing neuropathology in PD., Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson’s disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

The aim of this study was to compare fecal microbiome, plasma, fecal and urine metabolomes, and serum biochemistry of adult companion dogs according to body condition scores. Blood, serum/plasma, urine, and fecal samples were collected from 66 clinically healthy, adult companion dogs of either normal weight (NW), overweight (OW), or obese dogs (OB). analyses included fecal microbiome analyses via 16S ribosomal RNA gene amplicon; sequencing, nontargeted plasma, fecal, and urine metabolomics using liquid chromatography/gas chromatography-mass; spectrometry, and serum biochemistry for each dog. Few significant differences in serum biochemistry and fecal microbiome Operational Taxonomic Unit (OTU) were found between weight groups and there was high OTU variation between individual dogs. NW dogs had higher relative abundance of the genus Eubacterium (log-fold change 4.3, adjusted P value = .003) and lower relative abundance of the family Bifidobacteriaceae (log-fold change −3.6, adjusted P value = .02) compared to OB dogs. The microbiome of NW dogs had higher OTU richness compared with OB dogs. Metabolome analysis showed 185 plasma, 37 fecal, and 45 urine metabolites that significantly differed between NW and OW or OB dogs. There were notable significant differences in relative abundance of several plasma phospholipid moieties and fecal volatile fatty acids between weight phenotypes. The combinations of host and gut microbiota and metabolic shifts suggest a pattern that could help detection of early metabolic changes in overweight dogs before the development of obesity related disease. The results of this study support the need for continued investigation into sensitive measures of metabolic aberrancies in overweight dogs.

Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α–synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of ⍺-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of ⍺-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.

Neuroinvasive infections with mosquito-borne alphaviruses such as Western equine encephalitis virus (WEEV) can cause post-encephalitic parkinsonism. To understand the mechanisms underlying these neurological effects, we examined the capacity of WEEV to induce progressive neurodegeneration in outbred CD-1 mice following non-lethal encephalitic infection. Animals were experientally infected with recombinant WEEV expressing firefly luciferase or dsRed (RFP) reporters and the extent of viral replication was controlled using passive immunotherapy. WEEV spread along the neuronal axis from the olfactory bulb to the entorhinal cortex, hippocampus and basal midbrain by 4 days post infection (DPI). Infection caused activation of microglia and astrocytes, selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and neurobehavioral abnormalities. After 8 weeks, surviving mice displayed continued loss of dopamine neurons in the SNpc, lingering glial cell activation and gene expression profiles consistent with a neurodegenerative phenotype. Strikingly, prominent proteinase K-resistant protein aggregates were present in the the entorhinal cortex, hippocampus and basal midbrain that stained positively for phospho-serine129 α-synuclein (SNCA). These results indicate that WEEV may cause lasting neurological deficits through a severe neuroinflammatory response promoting both neuronal injury and protein aggregation in surviving individuals.

The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1+ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations., Author summary The rapid spread of Zika virus through a naïve population in the Americas resulted in novel and severe disease manifestations, including fetal and neonatal microcephaly, and GBS. These disease complications make understanding the pathology and ecology of ZIKV a priority. Captive Jamaican fruit bats were challenged with ZIKV to determine their susceptibility, to assess whether bats may play a role in virus ecology, and if they might serve as an animal model to better understand ZIKV pathophysiology. The bats became acutely infected and mounted an antibody response. Three terminally euthanized inoculated bats had antibody titers of 3200, 28 days PI. Evidence of virus replication and associated pathologies were found in the brain, testes, lungs and salivary glands of some of the inoculated bats. The virus showed predilection for mononuclear cells, including resident Iba1+ macrophage/microglial cells, and Leydig cells. With no discernible disruption to the blood brain barrier nor distribution of viral antigen indicative of circumeventricular neuroinvasion, microglia cells may be a possible route of entry of ZIKV into brains of bats. Further investigations are needed to determine the mechanisms of neuroinvasion of ZIKV in bats, further determine feasibility of bats as an alternative animal-model for congenital Zika syndrome, and what role bats might play in ZIKV viral ecology.