PI3K/AKT/mTOR Signaling Pathway Is Required for JCPyV Infection in Primary Astrocytes

Astrocytes are a main target of JC polyomavirus (JCPyV) in the central nervous system (CNS), where the destruction of these cells, along with oligodendrocytes, leads to the fatal disease progressive multifocal leukoencephalopathy (PML). There is no cure currently available for PML, so it is essential to discover antivirals for this aggressive disease. Additionally, the lack of a tractable in vivo models for studying JCPyV infection makes primary cells an accurate alternative for elucidating mechanisms of viral infection in the CNS. This research to better understand the signaling pathways activated in response to JCPyV infection reveals and establishes the importance of the PI3K/AKT/mTOR signaling pathway in JCPyV infection in primary human astrocytes compared to transformed cell lines. Using RNA sequencing and chemical inhibitors to target PI3K, AKT, and mTOR, we have demonstrated the importance of this signaling pathway in JCPyV infection of primary astrocytes not observed in transformed cells. Collectively, these findings illuminate the potential for repurposing drugs that are involved with inhibition of the PI3K/AKT/mTOR signaling pathway and cancer treatment as potential therapeutics for PML, caused by this neuroinvasive virus.

The Anti-diabetic Drug Gliquidone Modulates Lipopolysaccharide-Mediated Microglial Neuroinflammatory Responses by Inhibiting the NLRP3 Inflammasome

The sulfonylurea drug gliquidone is FDA approved for the treatment of type 2 diabetes. Binding of gliquidone to ATP-sensitive potassium channels (SUR1, Kir6 subunit) in pancreatic β-cells increases insulin release to regulate blood glucose levels. Diabetes has been associated with increased levels of neuroinflammation, and therefore the potential effects of gliquidone on micro- and astroglial neuroinflammatory responses in the brain are of interest. Here, we found that gliquidone suppressed LPS-mediated microgliosis, microglial hypertrophy, and proinflammatory cytokine COX-2 and IL-6 levels in wild-type mice, with smaller effects on astrogliosis. Importantly, gliquidone downregulated the LPS-induced microglial NLRP3 inflammasome and peripheral inflammation in wild-type mice. An investigation of the molecular mechanism of the effects of gliquidone on LPS-stimulated proinflammatory responses showed that in BV2 microglial cells, gliquidone significantly decreased LPS-induced proinflammatory cytokine levels and inhibited ERK/STAT3/NF-κB phosphorylation by altering NLRP3 inflammasome activation. In primary astrocytes, gliquidone selectively affected LPS-mediated proinflammatory cytokine expression and decreased STAT3/NF-κB signaling in an NLRP3-independent manner. These results indicate that gliquidone differentially modulates LPS-induced microglial and astroglial neuroinflammation in BV2 microglial cells, primary astrocytes, and a model of neuroinflammatory disease.

The MAPK/ERK Pathway and the Role of DUSP1 in JCPyV Infection of Primary Astrocytes

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates. In the present study, we tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes. AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1x10E5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1x10E4 viral genomes per cell) lead to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker, which may indicate functional alterations induced by viral transduction and/or transgene expression.

Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins—and of secreted metabolites—involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome–lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte–MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.

Attenuated Induction of the Unfolded Protein Response in Adult Human Primary Astrocytes in Response to Recurrent Low Glucose

Aims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure.MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq, respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform.Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following low glucose (LG) exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures.Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be a consequence of a successful metabolic adaptation, as previously reported, that better preserves intracellular energy levels and a reduced necessity for the UPR.

Studies of involvement of G-protein coupled receptor-3 in cannabidiol effects on inflammatory responses of mouse primary astrocytes and microglia

Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.