Epidermal Growth Factor Receptor-PI3K Signaling Controls Cofilin Activity To Facilitate Herpes Simplex Virus 1 Entry into Neuronal Cells

Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases

Cofilin-1 is involved in regulation of actin reorganization during influenza A virus assembly and budding

Influenza A virus (IAV) assembly and budding on host cell surface plasma membrane requires actin cytoskeleton reorganization. The underlying molecular mechanism involving actin reorganization remains unclarified. In this study, we found that the natural antiviral compound petagalloyl glucose (PGG) inhibits F-actin reorganization in the host cell membrane during the late stage of IAV infection, which are associated with the suppression of total cofilin-1 level and its phosphorylation. Knock-down of cofilin-1 reduces viral yields. These findings provide the first evidence that cofilin-1 plays an important role in regulating actin reorganization during IAV assembly and budding

Heat-Shock Protein 90 Promotes Nuclear Transport of Herpes Simplex Virus 1 Capsid Protein by Interacting with Acetylated Tubulin

Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of α-tubulin and Hsp90 interacted with the acetylated α-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated α-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance

Synthetic Analyses of Single-Cell Transcriptomes from Multiple Brain Organoids and Fetal Brain

Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future

Introductions to the Community: Early-Career Researchers in the Time of COVID-19

10.1016/j.stem.2020.10.008CELL STEM CELL275702-70

Ethanol Upregulates NMDA Receptor Subunit Gene Expression in Human Embryonic Stem Cell-Derived Cortical Neurons

<div><p>Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as <i>in vitro</i> cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (<i>ALDH2</i>), anti-apoptosis (<i>BCL2</i> and <i>CCND2</i>), neurotransmission (NMDA receptor subunit genes: <i>GRIN1</i>, <i>GRIN2A</i>, <i>GRIN2B</i>, and <i>GRIN2D</i>), calcium channel activity (<i>ITPR2</i>), or transcriptional repression (<i>JARID2</i>). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (<i>GRIN1</i>: 1.93-fold, <i>P</i> = 0.003; <i>GRIN2A</i>: 1.40-fold, <i>P</i> = 0.003; <i>GRIN2B</i>: 1.75-fold, <i>P</i> = 0.002; <i>GRIN2D</i>: 1.86-fold, <i>P</i> = 0.048; <i>BCL2</i>: 1.34-fold, <i>P</i> = 0.031), and the results of <i>GRIN1</i>, <i>GRIN2A</i>, and <i>GRIN2B</i> survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.</p></div

Effects of ethanol on gene expression in cortical neurons differentiated from H1 hESCs.

<p>Expression changes of nine genes in H1 hESC differentiated cortical neurons exposed to 50 mM ethanol for 7 days plus 24-hr ethanol withdrawal treatment were determined by RT-qPCR. Left column for each gene: without ethanol exposure; right column for each gene: with ethanol exposure. The expression level of each gene in ethanol-unexposed neurons was normalized to “1”. <i>GRIN1</i>, <i>2A</i>, <i>2B</i>, and <i>2D</i>: the NMDA receptor subunit genes; <i>BCL2</i>: the B-cell CLL/lymphoma 2 gene; <i>CCND2</i>: the cyclin D2 gene; <i>ALDH2</i>: the aldehyde dehydrogenase 2 gene; <i>ITPR2</i>: the inositol 1,4,5-trisphosphate receptor (type 2) gene; and <i>JARID2</i>: the jumonji- and AT-rich interactive domain 2 gene. The results were representative of three separate experiments. Each value represented the mean ± SD of triplicate wells.</p

Morphological changes of H1 hESC-derived cortical neurons exposed to ethanol.

<p>Morphological changes of H1 hESC-derived cortical neurons exposed to different concentrations of ethanol (0, 1, 10, 50, and 100 mM) were examined by DAPI DNA staining and Tuj1 (antibody specific for neuronal marker beta-III tubulin) immunostaining.</p