Morphological, Cellular, and Molecular Basis of Brain Infection in COVID-19 Patients and Its Association to Psychiatric Symptoms

Background: Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. Method(s): We performed a broad translational investigation, employing brain imaging and cognitive tests in 81 living COVID-19 patients (mildly infected individuals) as well as flow cytometry, respirometry, microscopy, proteomics, and metabolomics in postmortem brain samples, and in preclinical in vitro and ex vivo models. Result(s): We observed orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms in living individuals. Postmortem brain tissue from 26 individuals who died of COVID-19 revealed histopathological signs of brain damage. Five individuals out of the 26 exhibited foci of SARS- CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a non-canonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that significantly reduces neuronal viability. Conclusion(s): Our data support the model in which COVID-19 alter cortical thickness, promoting psychiatric symptoms. In addition, SARS-CoV-2 is able to reach the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients. Funding Source: Sao Paulo Research Foundation (FAPESP) Keywords: COVID-19, Anxiety, Astrocytes, Multi-omics, Brain Magnetic Resonance Imaging (MRI)Copyright © 2023

New antidepressants: monoamines and beyond

The monoamine hypothesis of depression has dominated treatment for decades, but for some with treatment-resistant depression, alternative approaches are needed. This article discusses some of the other mechanisms involved in depression and how novel treatments could address these.Copyright © 2023 Wiley Interface Ltd.

The relationship between the serotonergic system and COVID-19 disease: A review.

COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which led to a pandemic started in Wuhan, China, in 2019. The rapid spread of the disease in the world, unprecedented mortality rate, and lack of definitive treatment for the disease have led to a global effort to develop effective vaccines as well as new therapeutic interventions. Immune cells activation with excessive inflammation is an important pathophysiological feature of COVID-19 that may impair the various organs functions. Accordingly, these could cause dysfunction in the brain with some symptoms such as respiratory failure, headache, impaired consciousness, olfactory and taste disorders, and severe neurological disorders such as encephalitis. It was found that there is a two-way communication between the immune system and the nervous system through classical neurotransmitters, hormones, and cytokines. Among neurotransmitters, serotonin plays important roles in the immune system and in regulating inflammatory responses by central and peripheral mechanisms. This article aimed to review the two-way relationship between the immune and the nervous systems by focusing on the serotonergic system and the emerging COVID-19 disease.

Ivermectin: Evaluation of Efficacy and Safety in COVID-19

The search for an effective and safe COVID-19 therapy involves, among other things, assessment of efficacy of medicines already used for the treatment of other diseases, and having potential antiviral activity against SARSCoV-2. The relevance of the presented study stems from ambiguous data on the off-label use of the antiparasitic medicine ivermectin for the treatment of COVID-19 patients. The aim of the study was to analyse ivermectin efficacy and safety for COVID-19 treatment, as reflected in the scientific literature. Ivermectin, an antiparasitic medicine from the group of macrocyclic lactones produced by Streptomyces avermitilis, stimulates release of the inhibitory neurotransmitter gamma-aminobutyric acid, which leads to impaired transmission of nerve impulses, paralysis and death of parasites. The results of preclinical studies show ivermectin's inhibitory activity against a number of RNA and DNA viruses, including SARS-CoV-2. The results of ivermectin clinical studies are ambiguous: a number of studies demonstrated a positive effect on the condition of COVID-19 patients, however, there is currently no convincing evidence of the validity and efficacy of ivermectin use for the prevention and treatment of COVID-19 patients. The safety profile of ivermectin is relatively favourable. Large randomised controlled trials are needed to fully assess the feasibility of using ivermectin in COVID-19.

ENTERIC NEURONAL VASOACTIVE INTESTINAL PEPTIDE MEDIATES SARS-COV-2 ASSOCIATED DIARRHEA

Background: Diarrhea is present in up to 36.6% 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. The influence of ER stress and enteric neuronderived vasoactive intestinal peptide (VIP) on the expression of Na+/H+ exchanger 3 (NHE3), an important transporter that mediates intestinal Na+/fluid absorption, was further examined. Methods: SARS-CoV-2 propagated in Vero-E6 cells was used to infect Caco-2, a human colon epithelial cell line that expresses SARS-CoV-2 entry receptor ACE2. The expression of ER stress markers, phospho-PERK, Xbp1s, and DAMP proteins, was examined by Western blotting. Primary mouse enteric neurons were treated with a conditioned medium of Caco- 2 cells that were infected with SARS-CoV-2 or treated with tunicamycin. VIP expression by cultured enteric neurons was assessed by RT-qPCR, Western blotting, and ELISA. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s indicating increased ER stress. Infected Caco-2 cells secreted DAMP proteins, including HSP70 and calreticulin, as revealed by proteomic and Western analyses. The expression of VIP mRNA in enteric neurons was up-regulated after treatment with a conditioned medium of SARS-CoV-2- infected Caco-2 cells (Mock, 1 ± 0.0885;and SARS-CoV-2, 1.351 ± 0.020, P=.005). CD91, a receptor for HSP70 and calreticulin, is abundantly expressed in cultured mouse and human enteric neurons and was up-regulated by a conditioned medium of SARS-CoV-2-infected Caco-2 cells. Tunicamycin, an inducer of ER stress, also induced the secretion of HSP70 and calreticulin, mimicking SARS-CoV-2 infection. Moreover, co-culture of enteric neurons with tunicamycin-treated Caco-2 cells stimulated VIP production as determined by ELISA. Co-treatment of Caco-2 cells with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in the membrane expression of NHE3. Conclusions: Our findings demonstrate that SARS-CoV-2 infection of enterocytes leads to ER stress and the release of DAMPs that up-regulate the expression and release of VIP by enteric neurons. The presence of ER stress together with the secreted VIP, in turn, inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in the enterocytes. These data highlight epithelial-neuronal crosstalk in COVID-19 related diarrhea. (Figure Presented)

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.

Gut Microbiota Metabolites in Major Depressive Disorder-Deep Insights into Their Pathophysiological Role and Potential Translational Applications.

The gut microbiota is a complex and dynamic ecosystem essential for the proper functioning of the organism, affecting the health and disease status of the individuals. There is continuous and bidirectional communication between gut microbiota and the host, conforming to a unique entity known as "holobiont". Among these crosstalk mechanisms, the gut microbiota synthesizes a broad spectrum of bioactive compounds or metabolites which exert pleiotropic effects on the human organism. Many of these microbial metabolites can cross the blood-brain barrier (BBB) or have significant effects on the brain, playing a key role in the so-called microbiota-gut-brain axis. An altered microbiota-gut-brain (MGB) axis is a major characteristic of many neuropsychiatric disorders, including major depressive disorder (MDD). Significative differences between gut eubiosis and dysbiosis in mental disorders like MDD with their different metabolite composition and concentrations are being discussed. In the present review, the main microbial metabolites (short-chain fatty acids -SCFAs-, bile acids, amino acids, tryptophan -trp- derivatives, and more), their signaling pathways and functions will be summarized to explain part of MDD pathophysiology. Conclusions from promising translational approaches related to microbial metabolome will be addressed in more depth to discuss their possible clinical value in the management of MDD patients.

SARS-COV-2 INDUCES EPITHYELIAL-NEURONAL CROSSTALK STIMULATING VASOACTIVE INTESTINAL PEPTIDE RELEASE AS A POTENTIAL MECHANISM OF COVID-19-ASSOCIATED DIARRHEA

Background: Up to 36.6% of COVID-19 patients have diarrheal symptoms and 48.1% test positive for SARS-CoV-2 via stool test. The mechanism of SARS-CoV-2-associated diarrhea remains poorly understood. We hypothesize that crosstalk between enterocytes and the enteric nervous system (ENS) plays a critical role in the pathogenesis of COVID-19-associated diarrhea. We studied the effects of SARS-CoV-2 on induction of endoplasmic reticulum (ER) stress and release of Damage Associated Molecular Patterns (DAMPs), which act on enteric neurons and stimulate the production of neurotransmitters. The influence of ER stress and enteric neuron-derived vasoactive intestinal peptide (VIP) on the expression of electrolyte transporter Na+/H+ exchanger 3 (NHE3) was also examined. Methods: SARS-CoV-2 (2019-nCoV/USA-WA1/2020) was propagated in Vero-E6 cells. Caco-2, a human colon epithelial cell line, expresses the essential SARS-CoV-2 entry receptor ACE2 and was thus used for infection (MOI, ~0.01). We used Western blotting to assess the expression of ER stress (phospho-PERK and Xbp1s) and DAMP (HMGB1) markers at 48 hours post-infection. Primary mouse enteric neurons were co-cultured with Caco-2 cells, pre-treated for 24 hours with 2 μM tunicamycin to induce ER stress. Supernatants from enteric neurons were used to assess the expression of VIP by ELISA. Primary enteric neurons were treated with HMGB1 or ATP (another form of DAMPs), and the expression of c-FOS, a marker of neuronal activity, was determined by Western blotting and immunofluorescence staining. Results: We found that SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s. Compared to uninfected control, infected Caco-2 cells secreted HMGB1 into culture media, indicating epithelial production of DAMPs in response to SARS-CoV-2 infection. Tunicamycin was used to induce ER-stress and secretion of HMGB1 by Caco-2, mimicking SARS-CoV-2 infection. Importantly, enteric neurons co-cultured with tunicamycin-treated Caco-2 cells secreted significantly higher levels of VIP. Treating Caco-2 cells with tunicamycin or VIP on the basolateral side led to decreased surface NHE3 expression, suggesting a potential impairment of intestinal electrolyte/fluid absorption. More-over, HMGB1 and ATP both increased the expression of phospho-c-FOS in cultured enteric neurons, indicating DAMP-induced neuronal activation. Conclusions: Our findings demon-strate that enterocytes infected by SARS-CoV-2 release DAMPs with the capacity to induce VIP secretion by the enteric neurons, which in turn acts on enterocytes and inhibits apical localization of NHE3. These findings establish basic mechanisms relevant to diarrheal disease in COVID-19 patients and identify potential targets for the treatment of SARS-CoV-2 infection of the gastrointestinal tract.

Molecular mechanisms of lidocaine.

Lidocaine is an amide-class local anesthetic used clinically to inhibit pain sensations. Systemic administration of lidocaine has antinociceptive, antiarrhythmic, anti-inflammatory, and antithrombotic effects. Lidocaine exerts these effects under both acute and chronic pain conditions and acute respiratory distress syndrome through mechanisms that can be independent of its primary mechanism of action, sodium channel inhibition. Here we review the pathophysiological underpinnings of lidocaine's role as an anti-nociceptive, anti-inflammatory mediated by toll-like receptor (TLR) and nuclear factor kappa-ß (NF-kß) signalling pathways and downstream cytokine effectors high mobility group box 1 (HMGB1) and tumour necrosis factor-α (TNF-α).