Isolation and Enrichment of Major Primary Neuroglial Cells from Neonatal Mouse Brain.

The central nervous system (CNS) relies on the complex interaction of neuroglial cells to carry out vital physiological functions. To comprehensively understand the structural and functional interplay between these neuroglial cells, it is essential to establish an appropriate in vitro system that can be utilized for thorough investigation. Traditional protocols for establishing primary neuronal and mixed glial cultures from prenatal mice or neural stem cells require sacrificing pregnant mice and have the drawback of yielding only specific types of cells. Our current protocol overcomes these drawbacks by utilizing the brain from day-0 pups to isolate CNS resident neuroglial cells including astrocytes, microglia, oligodendrocytes [oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes], and meningeal fibroblasts, as well as hippocampal neurons, avoiding sacrificing pregnant mice, which makes this procedure efficient and cost effective. Furthermore, through this protocol, we aim to provide step-by-step instructions for isolating and establishing different primary neuroglial cells and their characterization using cell-specific markers. This study presents an opportunity to isolate, culture, and establish all major CNS resident cells individually. These cells can be utilized in various cell-based and biochemical assays to comprehensively investigate the cell-specific roles and behaviors of brain resident cells in a reductionist approach. Key features • Efficient isolation of major neuroglial cells like meningeal fibroblasts, neurons, astrocytes, oligodendrocytes, and microglia from a single day-0 neonatal mouse pup's brain. • Circumvents the sacrifice of pregnant female mice. • Acts as a bridging experimental method between secondary cell lines and in vivo systems. • Isolated cells can be used for performing various cell-based and biochemical assays.

Connexin 43 mediated collective cell migration is independent of Golgi orientation.

Cell migration is vital for multiple physiological functions and is involved in the metastatic dissemination of tumour cells in various cancers. For effective directional migration, cells often reorient their Golgi apparatus and, therefore, the secretory traffic towards the leading edge. However, not much is understood about the regulation of Golgi's reorientation. Herein, we address the role of gap junction protein Connexin 43 (Cx43), which connects cells, allowing the direct exchange of molecules. We utilized HeLa WT cells lacking Cx43 and HeLa 43 cells, stably expressing Cx43, and found that functional Cx43 channels affected Golgi morphology and reduced the reorientation of Golgi during cell migration. Although the migration velocity of the front was reduced in HeLa 43, the front displayed enhanced coherence in movement, implying an augmented collective nature of migration. On BFA treatment, Golgi was dispersed and the high heterogeneity in inter-regional front velocity of HeLa WT cells was reduced to resemble the HeLa 43. HeLa 43 had higher vimentin expression and stronger basal F-actin. Furthermore, non-invasive measurement of basal membrane height fluctuations revealed a lower membrane tension. We, therefore, propose that reorientation of Golgi is not the major determinant of migration in the presence of Cx43, which induces collective-like coherent migration in cells.

Azadirachta indica A. Juss bark extract and its Nimbin isomers restrict ß-coronaviral infection and replication.

Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-ß-Coronaviruses.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in murine ß-coronavirus-induced neuroinflammation.

Mouse hepatitis virus (MHV; m-ß-CoV) serves as a useful model for studying the cellular factors involved in neuroinflammation. To understand the role of matrix metalloproteinases (MMPs) in neuroinflammation, brain tissues from m-ß-CoV-infected mice were harvested at different days post-infection (d.p.i) and investigated for Mmp expression by RT-qPCR. Mmp-2, -3, -8, -12 showed significant mRNA upregulation peaking with viral replication between 5 and 6 d.p.i. Elevated levels of MMP regulator TIMP-1 are suggestive of a TIMP-1 mediated host antiviral response. Biological network assessment suggested a direct involvement of MMP-3, -8, -14 in facilitating peripheral leukocyte infiltrations. Flow cytometry confirmed the increased presence of NK cells, CD4+ and CD8+ T cells, neutrophils, and MHCII expressing cells in the m-ß-CoV infected mice brain. Our study revealed that m-ß-CoV upregulated Park7, RelA, Nrf2, and Hmox1 transcripts involved in ROS production and antioxidant pathways, describing the possible nexus between oxidative pathways, MMPs, and TIMP in m-ß-CoV-induced neuroinflammation.

DJ-1-Nrf2 axis is activated upon murine ß-coronavirus infection in the CNS.

Coronaviruses have emerged as alarming pathogens owing to their inherent ability of genetic variation and cross-species transmission. Coronavirus infection burdens the endoplasmic reticulum (ER.), causes reactive oxygen species production and induces host stress responses, including unfolded protein response (UPR) and antioxidant system. In this study, we have employed a neurotropic murine ß-coronavirus (M-CoV) infection in the Central Nervous System (CNS) of experimental mice model to study the role of host stress responses mediated by interplay of DJ-1 and XBP1. DJ-1 is an antioxidant molecule with established functions in neurodegeneration. However, its regulation in virus-induced cellular stress response is less explored. Our study showed that M-CoV infection activated the glial cells and induced antioxidant and UPR genes during the acute stage when the viral titer peaks. As the virus particles decreased and acute neuroinflammation diminished at day ten p.i., a significant up-regulation in UPR responsive XBP1, antioxidant DJ-1, and downstream signaling molecules, including Nrf2, was recorded in the brain tissues. Additionally, preliminary in silico analysis of the binding between the DJ-1 promoter and a positively charged groove of XBP1 is also investigated, thus hinting at a mechanism behind the upregulation of DJ-1 during MHV-infection. The current study thus attempts to elucidate a novel interplay between the antioxidant system and UPR in the outcome of coronavirus infection.

Spike Glycoprotein Is Central to Coronavirus Pathogenesis-Parallel Between m-CoV and SARS-CoV-2.

Background: Coronaviruses (CoVs) are single-stranded, polyadenylated, enveloped RNA of positive polarity with a unique potential to alter host tropism. This has been exceptionally demonstrated by the emergence of deadly virus outbreaks of the past: Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and Middle East Respiratory Syndrome (MERS-CoV) in 2012. Summary: The 2019 outbreak by the new cross-species transmission of SARS-CoV-2 has put the world on alert. CoV infection is triggered by receptor recognition, membrane fusion, and successive viral entry mediated by the surface Spike (S) glycoprotein. S protein is one of the major antigenic determinants and the target for neutralizing antibodies. It is a valuable target in antiviral therapies because of its central role in cell-cell fusion, viral antigen spread, and host immune responses leading to immunopathogenesis. The receptor-binding domain of S protein has received greater attention as it initiates host attachment and contains major antigenic determinants. However, investigating the therapeutic potential of fusion peptide as a part of the fusion core complex assembled by the heptad repeats 1 and 2 (HR1 and HR2) is also warranted. Along with receptor attachment and entry, fusion mechanisms should also be explored for designing inhibitors as a therapeutic intervention. Key message: In this article, we review the S protein function and its role in mediating membrane fusion, spread, tropism, and its associated pathogenesis with notable therapeutic strategies focusing on results obtained from studies on a murine ß-Coronavirus (m-CoV) and its associated disease process.

Connexin 43/47 channels are important for astrocyte/ oligodendrocyte cross-talk in myelination and demyelination.

The gap junctions (GJs), which form intercellular communicating channels between two apposing cells or form hemichannel with extracellular environment, perform crucial functions to maintain small molecule homeostasis. The central nervous system (CNS) GJs are important for maintenance of myelin sheath and neuronal activity. Connexin (Cx) proteins are building blocks of GJs. Recent cell-biological investigations show that amongst the CNS specific Cxs, the most abundant Cx protein, Cx43 and its oligodendrocytic coupling partner Cx47 primarily important for maintenance of CNS myelin. Recent investigations elucidate that the expression of Cx43 and Cx47 is very important to maintain K? buffering and nutrient homeostasis in oligodendrocytes, CNS myelin and oligodendrocyte function. The investigations on Multiple Sclerosis (MS) patient samples and EAE hypothesized that the functional loss of Cx43/Cx47 could be associated with spread of chronic MS lesions. Exploring the mechanism of initial GJ alteration and its effect on demyelination in this model of MS might play a primary role to understand the basis of altered CNS homeostasis, observed during MS. In this review, we mainly discuss the role of CNS GJs, specifically the Cx43/Cx47 axis in the perspective of demyelination.

Cytoplasmic tail of coronavirus spike protein has intracellular targeting signals.

Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spike protein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in the lysosome. Differential localization can be explained by signal sequence. The sequence alignment using Clustal W shows that the signal sequence present at the cytoplasmic tail plays an important role in spike protein localization. A unique GYQEL motif is identified at the cytoplasmic terminal of OC43 spike protein which helps in localization in the lysosome, and a novel KLHYT motif is identified in the cytoplasmic tail of SARS spike protein which helps in ER or ERGIC localization. This study sheds some light on the role of cytoplasmic tail of spike protein in cell-to-cell fusion, coronavirus host cell fusion and subsequent pathogenicity.

Mouse hepatitis virus infection upregulates genes involved in innate immune responses.

Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-γ, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-γ production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination.

ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum.

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.