Single-cell transcriptomic atlas of distinct early immune responses induced by SARS-CoV-2 Proto or its variants in rhesus monkey.

Immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection play a critical role in the pathogenesis and outcome of coronavirus disease 2019 (COVID-19). However, the dynamic profile of immune responses postinfection by SARS-CoV-2 variants of concern (VOC) is not fully understood. In this study, peripheral blood mononuclear cells single-cell sequencing was performed to determine dynamic profiles of immune response to Prototype, Alpha, Beta, and Delta in a rhesus monkey model. Overall, all strains induced dramatic changes in both cellular subpopulations and gene expression levels at 1 day postinfection (dpi), which associated function including adaptive immune response, innate immunity, and IFN response. COVID-19-related genes revealed different gene profiles at 1 dpi among the four SARS-CoV-2 strains, including genes reported in COVID-19 patients with increased risk of autoimmune disease and rheumatic diseases. Delta-infected animal showed inhibition of translation pathway. B cells, T cells, and monocytes showed much commonality rather than specificity among the four strains. Monocytes were the major responders to SARS-CoV-2 infection, and the response lasted longer in Alpha than the other strains. Thus, this study reveals the early immune responses induced by SARS-CoV-2 Proto or its variants in nonhuman primates, which is important information for controlling rapidly evolving viruses.

Nuclear localization of alpha-synuclein induces anxiety-like behavior in mice by decreasing hippocampal neurogenesis and pathologically affecting amygdala circuits.

Fear and anxiety are common in Parkinson's disease (PD) and may be caused by pathologies outside the dopaminergic system. Increasing evidence has shown that alpha-synuclein (α-syn) is involved in the development of anxiety in PD. In this study, we examined the effects of α-syn nuclear translocation on anxiety-like behavior in mice by overexpressing α-syn in the nuclei of the cell in the hippocampus. Our results show that α-syn overexpression in the nuclei increased the excitability of hippocampal neurons and activated NG2 glial cells and promoted the synthesis and release of γ-aminobutyric acid (GABA). And nuclear localization of α-syn led to the loss of neurotrophic factors and decreased neurogenesis. Meanwhile, the hippocampus and amygdala acted synergistically, resulting in pathologic accumulation of α-syn and gliosis in the amygdala and caused loss of interneurons. These events led to the impairments of hippocampus and amygdala function, which ultimately induced anxiety-like behavior in mice. The findings obtained in our present study indicate that excessive nuclear translocation of α-syn in hippocampal neurons and damage to the amygdala circuits may be important in the development of anxiety in PD.

Differential Transcriptomic Landscapes of SARS-CoV-2 Variants in Multiple Organs from Infected Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the persistent coronavirus disease 2019 (COVID-19) pandemic, which has resulted in millions of deaths worldwide and brought an enormous public health and global economic burden. The recurring global wave of infections has been exacerbated by growing variants of SARS-CoV-2. In this study, the virological characteristics of the original SARS-CoV-2 strain and its variants of concern (VOCs; including Alpha, Beta, and Delta) in vitro, as well as differential transcriptomic landscapes in multiple organs (lung, right ventricle, blood, cerebral cortex, and cerebellum) from the infected rhesus macaques, were elucidated. The original strain of SARS-CoV-2 caused a stronger innate immune response in host cells, and its VOCs markedly increased the levels of subgenomic RNAs, such as N, Orf9b, Orf6, and Orf7ab, which are known as the innate immune antagonists and the inhibitors of antiviral factors. Intriguingly, the original SARS-CoV-2 strain and Alpha variant induced larger alteration of RNA abundance in tissues of rhesus monkeys than Beta and Delta variants did. Moreover, a hyperinflammatory state and active immune response were shown in the right ventricles of rhesus monkeys by the up-regulation of inflammation- and immune-related RNAs. Furthermore, peripheral blood may mediate signaling transmission among tissues to coordinate the molecular changes in the infected individuals. Collectively, these data provide insights into the pathogenesis of COVID-19 at the early stage of infection by the original SARS-CoV-2 strain and its VOCs.

Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target.

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

Splicing factor SF3B3, a NS5-binding protein, restricts ZIKV infection by targeting GCH1.

Zika virus (ZIKV), a positive-sense single-stranded RNA virus, causes congenital ZIKV syndrome in children and Guillain-Barré Syndrome (GBS) in adults. ZIKV expresses nonstructural protein 5 (NS5), a large protein that is essential for viral replication. ZIKV NS5 confers the ability to evade interferon (IFN) signalling; however, the exact mechanism remains unclear. In this study, we employed affinity pull-down and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and found that splicing factor 3b subunit 3 (SF3B3) is associated with the NS5-Flag pull-down complex through interaction with NS5. Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene (ISG) expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication. GTP cyclohydrolase I (GCH1) is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis. NS5 upregulates the expression of GCH1 during ZIKV infection. And GCH1 marginally promoted ZIKV replication via the IFN pathway. Additionally, GCH1 expression is related to the regulation of SF3B3. Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels, whereas SF3B3 knockdown increased its levels. These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.

Nuclear localization of alpha-synuclein affects the cognitive and motor behavior of mice by inducing DNA damage and abnormal cell cycle of hippocampal neurons.

Nuclear accumulation of alpha-synuclein (α-syn) in neurons can promote neurotoxicity, which is considered the key factor in the pathogenesis of synucleinopathy. The damage to hippocampus neurons driven by α-syn pathology is also the potential cause of memory impairment in Parkinson's disease (PD) patients. In this study, we examined the role of α-syn nuclear translocation in the cognition and motor ability of mice by overexpressing α-syn in cell nuclei in the hippocampus. The results showed that the overexpression of α-syn in nuclei was able to cause significant pathological accumulation of α-syn in the hippocampus, and quickly lead to memory and motor impairments in mice. It might be that nuclear overexpression of α-syn may cause DNA damage of hippocampal neurons, thereby leading to activation and abnormal blocking of cell cycle, and further inducing apoptosis of hippocampal neurons and inflammatory reaction. Meanwhile, the inflammatory reaction further aggravated DNA damage and formed a vicious circle. Therefore, the excessive nuclear translocation of α-syn in hippocampal neurons may be one of the main reasons for cognitive decline in mice.

Absence of active systemic anaphylaxis in guinea pigs upon intramuscular injection of inactivated SARS-CoV-2 vaccine (Vero cells).

Background: The safety of novel vaccines against COVID-19 is currently a major focus of preclinical research. As a part of the safety evaluation testing package, 24 healthy guinea pigs were used to determine whether repeated administration of inactivated SARS-CoV-2 vaccine could induce active systemic anaphylaxis (ASA), and to evaluate its degree of severity.Method: According to sex and body weight, the animals were randomly divided into three experimental groups (eight animals per group). The negative control group received 0.9% sodium chloride (priming dose: 0.5 mL/animal; challenge dose: 1 mL/animal); the positive control group received 10% ovalbumin (priming dose: 0.5 mL/animal; challenge dose: 1 mL/animal); and the inactivated SARS-CoV-2 vaccine group received inactivated SARS-CoV-2 vaccines (priming dose: 100 U in 0.5 mL/animal; challenge dose: 200 U in 1 mL/animal). Priming dose administration was conducted by multi-point injection into the muscles of the hind limbs, three times, once every other day. On days 14 and 21 after the final priming injection, a challenge test was conducted. Half of the animals in each group were injected intravenously with twice the dose and volume of the tested substance used for immunization. During the experimental course, the injection site, general clinical symptoms, body weight, and systemic allergic reaction symptoms were monitored.Result: After intramuscular injection of inactivated SARS-CoV-2 vaccine, there were no abnormal reactions at the injection site, clinical symptoms, or deaths. There was no difference in body weight between the groups, and there were no allergic reactions. Conclusion: Thus, inactivated SARS-CoV-2 vaccine injected intramuscularly in guinea pigs did not produce ASA and had a good safety profile, which can provide actual data on vaccine risks and important reference data for clinical research on this vaccine.

Characteristic analysis of Omicron-included SARS-CoV-2 variants of concern.

In view of the rapid development of the COVID-19 pandemic and SARS-CoV-2 mutation, we characterized the emerging SARS-CoV-2 variants of concern (VOCs) by both bioinformatics methods and experiments. The representative genomic sequences of SARS-CoV-2 VOCs were first downloaded from NCBI, including the prototypic strain, Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Delta (B.1.617.2), and Omicron (B1.1.529) strain. Bioinformatics analysis revealed that the D614G mutation led to formation of a protruding spike (S) in the tertiary structure of spike protein, which could be responsible for the enhanced binding to angiotensin-converting enzyme 2 (ACE2) receptor. The epitope analysis further showed that the S protein antigenicity of the Omicron variant changed dramatically, which was possibly associated with its enhanced ability of immune escape. To verify the bioinformatics results, we performed experiments of pseudovirus infection and protein affinity assay. Notably, we found that the spike protein of Omicron variant showed the weakest infectivity and binding ability among all tested strains. Finally, we also proved this through virus infection experiments, and found that the cytotoxicity of Omicron seems to be not strong enough. The results in this study provide guidelines for prevention and control of COVID-19.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

Fat mass and obesity-associated protein regulates RNA methylation associated with depression-like behavior in mice.

Post-transcriptional modifications of RNA, such as RNA methylation, can epigenetically regulate behavior, for instance learning and memory. However, it is unclear whether RNA methylation plays a critical role in the pathophysiology of major depression disorder (MDD). Here, we report that expression of the fat mass and obesity associated gene (FTO), an RNA demethylase, is downregulated in the hippocampus of patients with MDD and mouse models of depression. Suppressing Fto expression in the mouse hippocampus results in depression-like behaviors in adult mice, whereas overexpression of FTO expression leads to rescue of the depression-like phenotype. Epitranscriptomic profiling of N6-methyladenosine (m6A) RNA methylation in the hippocampus of Fto knockdown (KD), Fto knockout (cKO), and FTO-overexpressing (OE) mice allows us to identify adrenoceptor beta 2 (Adrb2) mRNA as a target of FTO. ADRB2 stimulation rescues the depression-like behaviors in mice and spine loss induced by hippocampal Fto deficiency, possibly via the modulation of hippocampal SIRT1 expression by c-MYC. Our findings suggest that FTO is a regulator of a mechanism underlying depression-like behavior in mice.

Neurological complications and infection mechanism of SARS-COV-2.

Currently, SARS-CoV-2 has caused a global pandemic and threatened many lives. Although SARS-CoV-2 mainly causes respiratory diseases, growing data indicate that SARS-CoV-2 can also invade the central nervous system (CNS) and peripheral nervous system (PNS) causing multiple neurological diseases, such as encephalitis, encephalopathy, Guillain-Barré syndrome, meningitis, and skeletal muscular symptoms. Despite the increasing incidences of clinical neurological complications of SARS-CoV-2, the precise neuroinvasion mechanisms of SARS-CoV-2 have not been fully established. In this review, we primarily describe the clinical neurological complications associated with SARS-CoV-2 and discuss the potential mechanisms through which SARS-CoV-2 invades the brain based on the current evidence. Finally, we summarize the experimental models were used to study SARS-CoV-2 neuroinvasion. These data form the basis for studies on the significance of SARS-CoV-2 infection in the brain.

Distribution of the Alpha-Synuclein in the Brain and the Primary Organs of the Rhesus Monkey.

Previous studies have shown that abnormal aggregation of alpha-synuclein (α-syn) protein is a major trigger of neurodegenerative diseases. The expression level of α-syn in different brain regions and the disease-susceptible regions varies with the development of the disease. The expression pattern of the α-syn protein in mouse brain has been precisely described in the literature. Some studies have also reported the ubiquitous expression of the α-syn protein in the central and peripheral in nonhuman primates (NHPs). However, little is known about the expression pattern of α-syn in the brain or in the primary organs of NHPs. Here, we investigated the expression profile of α-syn in different brain regions and the primary organs of NHPs. The α-syn protein was mainly distributed in layers III and V of the cerebral cortex and the hippocampus. In addition, strong immunofluorescent signals were detected in the striatum and the substantia nigra, especially in the globus pallidus and the substantia nigra pars compacta, where the expression was significantly and particularly strong, compared with that in the cerebellum or the cortex. In the cerebellum, intense α-syn signal was observed in the molecular layer, where it was significantly higher than in the nucleus or the medulla. In the brain, the α-syn was always detected both in the cytoplasm and the synapses. Additionally, the α-syn was widely expressed in primary organs. The α-syn signal was higher in the liver and small intestine than in the spleen. Thus, the regions displaying the highest α-syn expression are also those affected during the progression of neurodegenerative diseases. These results may provide basic reference data for the study of multi-systemic mechanism of neurodegenerative diseases.

SARS-CoV-2 inactivated vaccine (Vero cells) shows good safety in repeated administration toxicity test of Sprague Dawley rats.

The outbreak of COVID-19 has posed a serious threat to global public health. Vaccination may be the most effective way to prevent and control the spread of the virus. The safety of vaccines is the focus of preclinical research, and the repeated dose toxicity test is the key safety test to evaluate the vaccine before clinical trials. The purpose of this study was (i) to observe the toxicity and severity of an inactivated SARS-CoV-2 vaccine (Vero cells) in rodent Sprague Dawley rats after multiple intramuscular injections under the premise of Good Laboratory Practice principles and (ii) to provide a basis for the formulation of a clinical trial scheme. The results showed that all animals in the experimental group were in good condition, no regular changes related to the vaccine were found in the detection of various toxicological indexes, and no noticeable stimulating reaction related to the vaccine was found in the injected local tissues. The neutralizing antibodies in the low- and high-dose vaccine groups began to appear 14 days after the last administration. In the negative control group, no neutralizing antibodies were observed from the administration period to the recovery period. Therefore, the repeated administration toxicity test of the inactivated SARS-CoV-2 vaccine (Vero cells) in Sprague Dawley rats showed no obvious toxic reaction. It was preliminarily confirmed that the vaccine can stimulate production of neutralizing antibodies and is safe in Sprague Dawley rats.

Hippocampal alpha-synuclein mediates depressive-like behaviors.

Alpha-synuclein (α-syn) which encoded by SNCA plays a critical role in the neurotransmission, vesicle dynamics, and neuroplasticity. Alteration to SNCA expression is associated with major depressive disorder. However, the pathogenic mechanism of SNCA in depression remains unknown. Herein, we reported that SNCA was up-regulated in the peripheral blood of major depressive disorder (MDD) patients and the depressive mice. Chronic restraint stress (CRS) also up-regulated the SNCA expression in the hippocampus. Moreover, over-expression of SNCA in the hippocampus triggered spontaneous depressive-like behaviors under the non-stressed conditions in mice, and knockout of SNCA could reverse CRS-induced depressive-like behaviors. SNCA led to synapse loss and neuronal cell death in the hippocampus possibly via complement-mediated microglial engulfment and inflammation, and thus contributed to the pathogenesis of depressive disorder. Overall, hippocampal SNCA and complement system are involved in the pathogenesis of depressive disorder and it provides a new perspective for the occurrence of depressive disorder.

Hsp90 inhibitor HSP990 in very low dose upregulates EAAT2 and exerts potent antiepileptic activity.

Rationale: Dysfunction or reduced levels of EAAT2 have been documented in epilepsy. We previously demonstrated the antiepileptic effects of Hsp90 inhibitor 17AAG in temporal lobe epilepsy by preventing EAAT2 degradation. Because of the potential toxicities of 17AAG, this study aimed to identify an alternative Hsp90 inhibitor with better performance on Hsp90 inhibition, improved blood-brain barrier penetration and minimal toxicity. Methods: We used cell-based screening and animal models of epilepsy, including mouse models of epilepsy and Alzheimer's disease, and a cynomolgus monkey model of epilepsy, to evaluate the antiepileptic effects of new Hsp90 inhibitors. Results: In both primary cultured astrocytes and normal mice, HSP990 enhanced EAAT2 levels at a lower dose than other Hsp90 inhibitors. In epileptic mice, administration of 0.1 mg/kg HSP990 led to upregulation of EAAT2 and inhibition of spontaneous seizures. Additionally, HSP990 inhibited seizures and improved cognitive functions in the APPswe/PS1dE9 transgenic model of Alzheimer's disease. In a cynomolgus monkey model of temporal lobe epilepsy, oral administration of low-dose HSP990 completely suppressed epileptiform discharges for up to 12 months, with no sign of hepatic and renal toxicity. Conclusions: These results support further preclinical studies of HSP990 treatment for temporal lobe epilepsy.

RNA demethylase Alkbh5 is widely expressed in neurons and decreased during brain development.

N6-methyladenosine (m6A) RNA methylation is one of the most abundant internal modifications on mRNAs and highly enriched within the brain. The demethylation of m6A is regulated by demethylases including fat-mass and obesity-associated protein (FTO) and AlkB homolog 5 (Alkbh5). FTO has been shown to play an important role in the brain, but little is known about the expression pattern and role of Alkbh5. Here, we investigated the expression profile of Alkbh5 in the developing mouse brain and its localization in the adult mouse brain. The results showed that Alkbh5 was widely detected throughout the mouse brain, with relatively high levels observed in the cerebellum and olfactory bulb of the adult mouse brain. Furthermore, Alkbh5 is mainly co-localized with neuronal marker NeuN, suggesting that it is primarily expressed in the neurons. Specifically, Alkbh5 could be found primarily in the nucleus of mouse neurons and cell lines. In addition, Alkbh5 protein decreased dramatically during brain development. Our findings detail the expression pattern and subcellular localization of Alkbh5 in the mouse brain. These results provide a neurobiological basis for the participation of Alkbh5 in the regulation of various brain functions, which might shed new light on further functional analysis of Alkbh5 and m6A in the central nervous system (CNS).

Injection of α-syn-98 Aggregates Into the Brain Triggers α-Synuclein Pathology and an Inflammatory Response.

Pathological aggregation of α-synuclein (α-syn) is a major component of Lewy bodies (LB), which play a central role in pathogenesis of Parkinson's disease (PD). Differential expression of α-syn isoforms has been shown in PD. Isoform α-syn-98 is generated by excision of exon-3 and exon-5 of the α-syn gene. In contrast to the canonical full-length α-syn isoform (α-syn140), little is known about the function of the α-syn-98 isoform. In the present study, to identify the potential role of α-syn-98 protein in PD, we examined the effects of exogenous recombinant insoluble α-syn-98 aggregates on α-syn pathology and inflammatory responses in the midbrain. After injection of α-syn-98 aggregates into the substantia nigra (SN), mice exhibited motor dysfunction accompanied by nigral dopaminergic neuron loss. In addition, α-syn-98 aggregates injection resulted in a significant increase in phosphorylation of endogenous α-syn. Accumulations of α-syn were co-localized with p62 and ubiquitin, which suggests α-syn-98 aggregates-induced pathology exhibits properties similar to human LB. Many glial cells were activated after α-syn-98 aggregates injection. In addition, expression of NF-κB, interleukin 6 (IL6), and tumor necrosis factor-α (TNF-α) and levels of oxidative stress increased after α-syn-98 aggregates injection. Our results suggest that α-syn-98 may play a crucial role in the pathogenesis of PD.

Alpha-synuclein is highly prone to distribution in the hippocampus and midbrain in tree shrews, and its fibrils seed Lewy body-like pathology in primary neurons.

The Chinese tree shrew (TS) has many unique advantages that make it suitable for use as an experimental animal model for human disease including moderate body size, low cost of feeding, short reproductive cycle and lifespan, and close phylogenetic relationship to primates. Our previous studies have shown that TS treated with the mitochondrial inhibitor MPTP displayed classic Parkinsonian symptoms. Additionally, the structure of TS alpha-synuclein (α-syn) is highly homologous to that found in humans. Previous studies have concluded that misfolded, fibrillar α-syn is a hallmark of α-synucleinopathies. In this study, we examined the distribution and expression levels of α-syn in different TS brain regions. We also obtained recombinant TS α-syn protein to study its aggregation and cytotoxic properties in primary neurons. Our results showed that α-syn was expressed in numerous different brain regions in TS but was most abundant in the hippocampus and midbrain. The recombinant α-syn of TS displayed straight fibrils when incubated for 72 h in vitro, which is very similar to human α-syn. When exposed to primary neurons, the TS and human α-syn fibrils led to cytotoxicity and Lewy-like pathology. Our findings indicated that TS could be a potential animal model to study the pathology of α-synucleinopathies.

Intergenic variants may predispose to major depression disorder through regulation of long non-coding RNA expression.

Genome-wide association (GWA) studies have identified numerous genetic variants for major depressive disorder (MDD) although most of the genetic variants are intergenic. It has been found that approximately 54% of long non-coding RNAs (lncRNAs) are located in the intergenic regions. We hypothesized that intergenic variants might be involved in the pathogenesis of MDD through regulating the expression of lncRNAs where these variants are located. In this study, several MDD-associated SNPs in three known intergenic lncRNAs were initially genotyped among 978 patients with MDD and 1176 controls, and the real-time PCR assay was performed to quantify the expression of LINC01108 and LINC00578 in peripheral blood cells from 20 MDD patients and 20 controls. The results showed that rs12526133 present in LINC01108 was strongly associated with MDD (χ2=11.68, P=6.3E-04), and LINC01108 expression was significantly higher in the patient group than in the control group (FC=1.90, P<0.001). The expression of LINC00998 was significantly lower in MDD patients than controls based on microarray analysis (FC=0.11, P<0.001), so that its tag SNPs were genotyped and rs2272260 in LINC00998 was found to be associated with MDD (χ2=26.39, P=2.8E-07). This work suggests that non-coding variants may play an important role in conferring risk of MDD.