Astrocyte inflammatory signaling mediates α-synuclein aggregation and dopaminergic neuronal loss following viral encephalitis.

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.

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for spillback to New World rodents.

Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNß, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.

DOG1 is a sensitive and specific immunohistochemical marker for diagnosis of canine gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) and leiomyosarcomas are histologically similar primary neoplasms commonly occurring in the gastrointestinal tract of dogs and humans. Immunohistochemical staining (IHC) is needed to differentiate between these 2 entities and positive reactivity for KIT (cluster of differentiation [CD]117) is regarded as the gold standard for diagnosis of canine GIST. Studies estimate 5-10% of human GISTs stain negative or only weakly positive for KIT and have identified DOG1 (discovered on gastrointestinal stromal tumors protein 1) as a highly sensitive and specific marker for human GISTs. The purpose of this study was to evaluate immunoreactivity of a commercially available DOG1 antibody for use in diagnosis of canine GISTs. Fifty-five primary mesenchymal gastrointestinal tumors with histologic features consistent with GIST or leiomyosarcoma were evaluated via IHC for KIT, DOG1, and desmin. A subset of tumors was additionally evaluated for reactivity for smooth muscle actin (SMA). Thirty-three tumors (60%) were diagnosed as GIST based on positive immunoreactivity for KIT or DOG1 regardless of reactivity for desmin or SMA. Most GISTs (32/33, 97.0%) had similar staining for both KIT and DOG1. DOG1 expression was identified in 2 tumors (1 study tumor and 1 additional tumor) negative for KIT and desmin that had histologic features consistent with KIT-negative, platelet-derived growth factor receptor-alpha (PDGFRA)-mutant human GISTs. Our results suggest that DOG1 has improved specificity and sensitivity to that of KIT for differentiating between canine GISTs and leiomyosarcomas. Inclusion of both DOG1 and KIT IHC in diagnostic panels will improve the accuracy of canine GIST diagnosis.

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.