SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

Loss of neuron network coherence induced by virus-infected astrocytes: a model study.

Coherent activations of brain neuron networks underlie many physiological functions associated with various behavioral states. These synchronous fluctuations in the electrical activity of the brain are also referred to as brain rhythms. At the cellular level, rhythmicity can be induced by various mechanisms of intrinsic oscillations in neurons or the network circulation of excitation between synaptically coupled neurons. One specific mechanism concerns the activity of brain astrocytes that accompany neurons and can coherently modulate synaptic contacts of neighboring neurons, synchronizing their activity. Recent studies have shown that coronavirus infection (Covid-19), which enters the central nervous system and infects astrocytes, can cause various metabolic disorders. Specifically, Covid-19 can depress the synthesis of astrocytic glutamate and gamma-aminobutyric acid. It is also known that in the post-Covid state, patients may suffer from symptoms of anxiety and impaired cognitive functions. We propose a mathematical model of a spiking neuron network accompanied by astrocytes capable of generating quasi-synchronous rhythmic bursting discharges. The model predicts that if the release of glutamate is depressed, normal burst rhythmicity will suffer dramatically. Interestingly, in some cases, the failure of network coherence may be intermittent, with intervals of normal rhythmicity, or the synchronization can disappear.

A functional role for alpha-synuclein in neuroimmune responses.

Alpha-synuclein is a neuronal protein with unclear function but is associated with the pathogenesis of Parkinson's disease and other synucleinopathies. In this review, we discuss the emerging functional role of alpha-synuclein in support of the unique immune responses in the nervous system. Recent data now show that alpha-synuclein functions to support interferon signaling within neurons and is released from neurons to support chemoattraction and activation of local glial cells and infiltrating immune cells. Inflammatory activation and interferon signaling also induce post-translational modifications of alpha-synuclein that are commonly associated with Parkinson's disease pathogenesis. Taken together, emerging data implicate complex interactions between alpha-synuclein and host immune responses that may contribute to the pathogenesis of Parkinson's disease. Additional study of the function of alpha-synuclein in the brain's immune response may provide disease-modifying therapeutic targets for Parkinson's disease in the future.

Transient Changes in the Plasma of Astrocytic and Neuronal Injury Biomarkers in COVID-19 Patients without Neurological Syndromes.

The levels of several glial and neuronal plasma biomarkers have been found to increase during the acute phase in COVID-19 patients with neurological symptoms. However, replications in patients with minor or non-neurological symptoms are needed to understand their potential as indicators of CNS injury or vulnerability. Plasma levels of glial fibrillary acidic protein (GFAP), neurofilament light chain protein (NfL), and total Tau (T-tau) were determined by Single molecule array (Simoa) immunoassays in 45 samples from COVID-19 patients in the acute phase of infection [moderate (n = 35), or severe (n = 10)] with minor or non-neurological symptoms; in 26 samples from fully recovered patients after ~2 months of clinical follow-up [moderate (n = 23), or severe (n = 3)]; and in 14 non-infected controls. Plasma levels of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), were also determined by Western blot. Patients with COVID-19 without substantial neurological symptoms had significantly higher plasma concentrations of GFAP, a marker of astrocytic activation/injury, and of NfL and T-tau, markers of axonal damage and neuronal degeneration, compared with controls. All these biomarkers were correlated in COVID-19 patients at the acute phase. Plasma GFAP, NfL and T-tau levels were all normalized after recovery. Recovery was also observed in the return to normal values of the quotient between the ACE2 fragment and circulating full-length species, following the change noticed in the acute phase of infection. None of these biomarkers displayed differences in plasma samples at the acute phase or recovery when the COVID-19 subjects were sub-grouped according to occurrence of minor symptoms at re-evaluation 3 months after the acute episode (so called post-COVID or "long COVID"), such as asthenia, myalgia/arthralgia, anosmia/ageusia, vision impairment, headache or memory loss. Our study demonstrated altered plasma GFAP, NfL and T-tau levels in COVID-19 patients without substantial neurological manifestation at the acute phase of the disease, providing a suitable indication of CNS vulnerability; but these biomarkers fail to predict the occurrence of delayed minor neurological symptoms.

SARS-CoV-2 Induces Epithelial-Enteric Neuronal Crosstalk Stimulating VIP Release.

BACKGROUND: Diarrhea is present in up to 30-50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. METHODS: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. RESULTS: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. CONCLUSIONS: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.

Wilful pathogens provoke a gut feeling in Parkinson's disease.

Parkinson's disease is the second most common neurological disorder marked by characteristic poverty and dysfunction in movement. There are many mechanisms and factors which have been postulated to be associated with the neurodegenerative pathway(s) resulting in distinctive loss of neurons in the substantia nigra. Subsequently, the neuropathology is more widespread and exhibited in other areas of the brain, and enteric nervous system. Aggregates of misfolded α-synuclein or Lewy bodies are the hallmark of the illness and appear to be central in the whole cascade of cell destruction. There are many processes implicated in neuronal destruction including: oxidative stress, excitotoxicity, mitochondrial dysfunction, an imbalance in protein homeostasis and neuroinflammation. Interesting, inflammation induced by pathogens (including, bacteria and viruses) has been associated in the pathogenesis of the disease. Bacteria such as Helicobacter pylori and Helicobacter suis appear to colonise the gut, and elicit systemic immune responses, which is them transmitted via the gut-axis to the brain, where cytotoxic cytokines induce neuroinflammation and cell death. This conforms to the bottom-top hypothesis proposed by Braak. The gut is also implicated in two other theories postulated in the development and progression of the disorder, namely, the top-down and the threshold. There is a possibility that these theories may be inter-linked and operate together to certain degree. Ultimately specific trigger factors or conditions may govern the occurrences of these processes in genetically predisposed individuals. Nevertheless, the importance of pathogen-related gut infections cannot be overlooked, since it can result in dysbiosis of gut microbes, which may orchestrate α-synuclein pathology and eventually cell destruction.

Special Issue Editorial: "Infections, Inflammation and Neurodegeneration in Alzheimer Disease" Infections, Neuronal Senescence, and Dementia.

Alzheimer's disease (AD) is a complex chronic disease of the brain characterized by several neurodegenerative mechanisms and is responsible for most dementia cases in the elderly. Declining immunity during ageing is often associated with peripheral chronic inflammation, and chronic neuroinflammation is a constant component of AD brain pathology. In the Special Issue published in 2021 eight papers were collected regarding different aspects of neurodegeneration associated with AD. Five papers presented and discussed infectious agents involved in brain AD pathology and three discussed data regarding receptors regulation and possible treatment of the disease. Below I will discuss and further elaborate on topics related to infections, inflammation, and neurodegenerative pathways in AD and brain senescence. The topic presented here may contribute to early intervention protocols for preventing or slowing the progression of cognitive deterioration in the elderly.

Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development.

The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).

Autism genes converge on asynchronous development of shared neuron classes.

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol.

The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+. The possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV.

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes.

Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.

Rh-CXCL-12 Attenuates Neuronal Pyroptosis after Subarachnoid Hemorrhage in Rats via Regulating the CXCR4/NLRP1 Pathway.

Subarachnoid hemorrhage (SAH) is a cerebrovascular disease associated with high morbidity and mortality. CXCR4 provides neuroprotective effects, which can alleviate brain injury and inflammation induced by stroke. Previous studies have suggested that CXCR4 reduces the pyroptosis of LPS-stimulated BV2 cells. The purpose of this study was to evaluate the antipyroptosis effects and mechanisms of CXCR4 after SAH. SAH animal model was induced via endovascular perforation. A total of 136 male Sprague-Dawley rats were used. Recombinant human cysteine-X-cysteine chemokine ligand 12 (rh-CXCL-12) was administered intranasally at 1 h after SAH induction. To investigate the underlying mechanism, the inhibitor of CXCR4, AMD3100, was administered intraperitoneally at 1 h before SAH. The neurobehavior tests were assessed, followed by performing Western blot and immunofluorescence staining. The Western blot results suggested that the expressions of endogenous CXCL-12, CXCR4, and NLRP1 were increased and peaked at 24 h following SAH. Immunofluorescence staining showed that CXCR4 was expressed on neurons, microglia, and astrocytes. Rh-CXCL-12 treatment improved the neurological deficits and reduced the number of FJC-positive cells, IL-18-positive neurons, and cleaved caspase-1(CC-1)-positive neurons after SAH. Meanwhile, rh-CXCL-12 treatment increased the levels of CXCL-12 and CXCR4, and reduced the levels of NLRP1, IL-18, IL-1ß, and CC-1. Moreover, the administration of AMD3100 abolished antipyroptosis effects of CXCL-12 and its regulation of CXCR4 post-SAH. The CXCR4/NLRP1 signaling pathway may be involved in CXCL-12-mediated neuronal pyroptosis after SAH. Early administration of CXCL-12 may be a preventive and therapeutic strategy against brain injury after SAH.

PDZ-Containing Proteins Targeted by the ACE2 Receptor.

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants' values ranging from 3 to 81 µM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

Porcine Hemagglutinating Encephalomyelitis Virus Triggers Neural Autophagy Independently of ULK1.

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (ß-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing ULK1-knockout neuroblastoma cells, we have identified that ULK1 is not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of noncanonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic ß-CoV and the host. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic alongside the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) pose Betacoronavirus (ß-CoV) as a global public health challenge. Coronaviruses subvert, hijack, or utilize autophagy to promote proliferation, and thus, exploring the cross talk between ß-CoV and autophagy is of great significance in confronting future ß-CoV outbreaks. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic ß-CoV that invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction is yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypasses the multifaceted regulation of ULK1 kinase. The PHEV-triggered noncanonical autophagy underscores the complex interactions of virus and host and will help in the development of therapeutic strategies targeting noncanonical autophagy to treat ß-CoV disease.

Expression of SARS-CoV-2-related receptors in cells of the neurovascular unit: implications for HIV-1 infection.

BACKGROUND: Neurological complications are common in patients affected by COVID-19 due to the ability of SARS-CoV-2 to infect brains. While the mechanisms of this process are not fully understood, it has been proposed that SARS-CoV-2 can infect the cells of the neurovascular unit (NVU), which form the blood-brain barrier (BBB). The aim of the current study was to analyze the expression pattern of the main SARS-CoV-2 receptors in naïve and HIV-1-infected cells of the NVU in order to elucidate a possible pathway of the virus entry into the brain and a potential modulatory impact of HIV-1 in this process. METHODS: The gene and protein expression profile of ACE2, TMPRSS2, ADAM17, BSG, DPP4, AGTR2, ANPEP, cathepsin B, and cathepsin L was assessed by qPCR, immunoblotting, and immunostaining, respectively. In addition, we investigated if brain endothelial cells can be affected by the exposure to the S1 subunit of the S protein, the domain responsible for the direct binding of SARS-CoV-2 to the ACE2 receptors. RESULTS: The receptors involved in SARS-CoV-2 infection are co-expressed in the cells of the NVU, especially in astrocytes and microglial cells. These receptors are functionally active as exposure of endothelial cells to the SARS CoV-2 S1 protein subunit altered the expression pattern of tight junction proteins, such as claudin-5 and ZO-1. Additionally, HIV-1 infection upregulated ACE2 and TMPRSS2 expression in brain astrocytes and microglia cells. CONCLUSIONS: These findings provide key insight into SARS-CoV-2 recognition by cells of the NVU and may help to develop possible treatment of CNS complications of COVID-19.

Neurochemical signs of astrocytic and neuronal injury in acute COVID-19 normalizes during long-term follow-up.

BACKGROUND: Neurologic manifestations are well-recognized features of coronavirus disease 2019 (COVID-19). However, the longitudinal association of biomarkers reflecting CNS impact and neurological symptoms is not known. We sought to determine whether plasma biomarkers of CNS injury were associated with neurologic sequelae after COVID-19. METHODS: Patients with confirmed acute COVID-19 were studied prospectively. Neurological symptoms were recorded during the acute phase of the disease and at six months follow-up, and blood samples were collected longitudinally. Healthy age-matched individuals were included as controls. We analysed plasma concentrations of neurofilament light-chain (NfL), glial fibrillary acidic protein (GFAp), and growth differentiation factor 15 (GDF-15). FINDINGS: One hundred patients with mild (n = 24), moderate (n = 28), and severe (n = 48) COVID-19 were followed for a median (IQR) of 225 (187-262) days. In the acute phase, patients with severe COVID-19 had higher concentrations of NfL than all other groups (all p < 0·001), and higher GFAp than controls (p < 0·001). GFAp was also significantly increased in moderate disease (p < 0·05) compared with controls. NfL (r = 0·53, p < 0·001) and GFAp (r = 0·39, p < 0·001) correlated with GDF-15 during the acute phase. After six months, NfL and GFAp concentrations had normalized, with no persisting group differences. Despite this, 50 patients reported persistent neurological symptoms, most commonly fatigue (n = 40), "brain-fog" (n = 29), and changes in cognition (n = 25). We found no correlation between persistent neurological symptoms and CNS injury biomarkers in the acute phase. INTERPRETATION: The normalization of CNS injury biomarkers in all individuals, regardless of previous disease severity or persisting neurological symptoms, indicates that post COVID-19 neurological sequelae are not accompanied by ongoing CNS injury. FUNDING: The Swedish State Support for Clinical Research, SciLifeLab Sweden, and the Knut and Alice Wallenberg Foundation have provided funding for this project.

Genetic Exchange of Lung-Derived Exosome to Brain Causing Neuronal Changes on COVID-19 Infection.

The pandemic of novel coronavirus 2 (SARS-CoV-2) has made global chaos for normal human living. Despite common COVID-19 symptoms, variability in clinical phenotypes was reported worldwide. Reports on SARS-CoV-2 suggest causing neurological manifestation. In addition, the susceptibility of SARS-CoV-2 in patients with neurodegenerative diseases and its complexity are largely unclear. Here, we aimed to demonstrate the possible transport of exosome from SARS-CoV-2-infected lungs to the brain regions associated with neurodegenerative diseases using multiple transcriptome datasets of SARS-CoV-2-infected lungs, RNA profiles from lung exosome, and gene expression profiles of the human brain. Upon transport, the transcription factors localized in the exosome regulate genes at lateral substantia nigra, medial substantia nigra, and superior frontal gyrus regions of Parkinson's disease (PD) and frontal cortex, hippocampus, and temporal cortex of Alzheimer's disease (AD). On SARS-CoV-2 infection, BCL3, JUND, MXD1, IRF2, IRF9, and STAT1 transcription factors in the exosomes influence the neuronal gene regulatory network and accelerate neurodegeneration. STAT1 transcription factor regulates 64 PD genes at lateral substantia nigra, 65 at superior frontal gyrus, and 19 at medial substantia nigra. Similarly, in AD, STAT1 regulates 74 AD genes at the temporal cortex, 40 genes at the hippocampus, and 16 genes at the frontal cortex. We further demonstrate that dysregulated neuronal genes showed involvement in immune response, signal transduction, apoptosis, and stress response process. In conclusion, SARS-CoV-2 may dysregulate neuronal gene regulatory network through exosomes that attenuate disease severity of neurodegeneration.

SARS-CoV-2 Neuronal Invasion and Complications: Potential Mechanisms and Therapeutic Approaches.

Clinical reports suggest that the coronavirus disease-19 (COVID-19) pandemic caused by severe acute respiratory syndrome (SARS)-coronavirus-2 (CoV-2) has not only taken millions of lives, but has also created a major crisis of neurologic complications that persist even after recovery from the disease. Autopsies of patients confirm the presence of the coronaviruses in the CNS, especially in the brain. The invasion and transmission of SARS-CoV-2 in the CNS is not clearly defined, but, because the endocytic pathway has become an important target for the development of therapeutic strategies for COVID-19, it is necessary to understand endocytic processes in the CNS. In addition, mitochondria and mechanistic target of rapamycin (mTOR) signaling pathways play a critical role in the antiviral immune response, and may also be critical for endocytic activity. Furthermore, dysfunctions of mitochondria and mTOR signaling pathways have been associated with some high-risk conditions such as diabetes and immunodeficiency for developing severe complications observed in COVID-19 patients. However, the role of these pathways in SARS-CoV-2 infection and spread are largely unknown. In this review, we discuss the potential mechanisms of SARS-CoV-2 entry into the CNS and how mitochondria and mTOR pathways might regulate endocytic vesicle-mitochondria interactions and dynamics during SARS-CoV-2 infection. The mechanisms that plausibly account for severe neurologic complications with COVID-19 and potential treatments with Food and Drug Administration-approved drugs targeting mitochondria and the mTOR pathways are also addressed.

IL-6 Inhibition Reduces Neuronal Injury in a Murine Model of Ventilator-induced Lung Injury.

Mechanical ventilation is a known risk factor for delirium, a cognitive impairment characterized by dysfunction of the frontal cortex and hippocampus. Although IL-6 is upregulated in mechanical ventilation-induced lung injury (VILI) and may contribute to delirium, it is not known whether the inhibition of systemic IL-6 mitigates delirium-relevant neuropathology. To histologically define neuropathological effects of IL-6 inhibition in an experimental VILI model, VILI was simulated in anesthetized adult mice using a 35 cc/kg tidal volume mechanical ventilation model. There were two control groups, as follow: 1) spontaneously breathing or 2) anesthetized and mechanically ventilated with 10 cc/kg tidal volume to distinguish effects of anesthesia from VILI. Two hours before inducing VILI, mice were treated with either anti-IL-6 antibody, anti-IL-6 receptor antibody, or saline. Neuronal injury, stress, and inflammation were assessed using immunohistochemistry. CC3 (cleaved caspase-3), a neuronal apoptosis marker, was significantly increased in the frontal (P < 0.001) and hippocampal (P < 0.0001) brain regions and accompanied by significant increases in c-Fos and heat shock protein-90 in the frontal cortices of VILI mice compared with control mice (P < 0.001). These findings were not related to cerebral hypoxia, and there was no evidence of irreversible neuronal death. Frontal and hippocampal neuronal CC3 were significantly reduced with anti-IL-6 antibody (P < 0.01 and P < 0.0001, respectively) and anti-IL-6 receptor antibody (P < 0.05 and P < 0.0001, respectively) compared with saline VILI mice. In summary, VILI induces potentially reversible neuronal injury and inflammation in the frontal cortex and hippocampus, which is mitigated with systemic IL-6 inhibition. These data suggest a potentially novel neuroprotective role of systemic IL-6 inhibition that justifies further investigation.