SARS-CoV-2 Spike protein alters microglial purinergic signaling.

Despite long-term sequelae of COVID-19 are emerging as a substantial public health concern, the mechanism underlying these processes still unclear. Evidence demonstrates that SARS-CoV-2 Spike protein can reach different brain regions, irrespective of viral brain replication resulting in activation of pattern recognition receptors (PRRs) and neuroinflammation. Considering that microglia dysfunction, which is regulated by a whole array of purinergic receptors, may be a central event in COVID-19 neuropathology, we investigated the impact of SARS-CoV-2 Spike protein on microglial purinergic signaling. Here, we demonstrate that cultured microglial cells (BV2 line) exposed to Spike protein induce ATP secretion and upregulation of P2Y6, P2Y12, NTPDase2 and NTPDase3 transcripts. Also, immunocytochemistry analysis shows that spike protein increases the expression of P2X7, P2Y1, P2Y6, and P2Y12 in BV2 cells. Additional, hippocampal tissue of Spike infused animals (6,5ug/site, i.c.v.) presents increased mRNA levels of P2X7, P2Y1, P2Y6, P2Y12, NTPDase1, and NTPDase2. Immunohistochemistry experiments confirmed high expression of the P2X7 receptor in microglial cells in CA3/DG hippocampal regions after spike infusion. These findings suggest that SARS-CoV-2 Spike protein modulates microglial purinergic signaling and opens new avenues for investigating the potential of purinergic receptors to mitigate COVID-19 consequences.

SARS-CoV-2 Spike protein induces TLR4-mediated long-term cognitive dysfunction recapitulating post-COVID-19 syndrome in mice.

Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents.

Nanosystems for gene therapy targeting brain damage caused by viral infections.

Several human pathogens can cause long-lasting neurological damage. Despite the increasing clinical knowledge about these conditions, most still lack efficient therapeutic interventions. Gene therapy (GT) approaches comprise strategies to modify or adjust the expression or function of a gene, thus providing therapy for human diseases. Since recombinant nucleic acids used in GT have physicochemical limitations and can fail to reach the desired tissue, viral and non-viral vectors are applied to mediate gene delivery. Although viral vectors are associated to high levels of transfection, non-viral vectors are safer and have been further explored. Different types of nanosystems consisting of lipids, polymeric and inorganic materials are applied as non-viral vectors. In this review, we discuss potential targets for GT intervention in order to prevent neurological damage associated to infectious diseases as well as the role of nanosized non-viral vectors as agents to help the selective delivery of these gene-modifying molecules. Application of non-viral vectors for delivery of GT effectors comprise a promising alternative to treat brain inflammation induced by viral infections.

Prion protein complexed to a DNA aptamer induce behavioral and synapse dysfunction in mice.

Conversion of the cellular prion protein (PrPC) into the scrapie form (PrPSc) is the leading step to the development of transmissible spongiform encephalopathies (TSEs), still incurable neurodegenerative disorders. Interaction of PrPC with cellular and synthetic ligands that induce formation of scrapie-like conformations has been deeply investigated in vitro. Different nucleic acid (NA) sequences bind PrP and convert it to ß-sheet-rich or unfolded species; among such NAs, a 21-mer double-stranded DNA, D67, was shown to induce formation of PrP aggregates that were cytotoxic. However, in vivo effects of these PrP-DNA complexes were not explored. Herein, aggregates of recombinant full-length PrP (rPrP23-231) induced by interaction with the D67 aptamer were inoculated into the lateral ventricle of Swiss mice and acute effects were investigated. The aggregates had no influence on emotional, locomotor and motor behavior of mice. In contrast, mice developed cognitive impairment and hippocampal synapse loss, which was accompanied by intense activation of glial cells in this brain region. Our results suggest that the i.c.v. injection of rPrP:D67 aggregates is an interesting model to study the neurotoxicity of aggregated PrP in vivo, and that glial cell activation may be an important step for behavioral and cognitive dysfunction in prion diseases.

Dissociation of genotype-dependent cognitive and motor behavior in a strain of aging mice devoid of the prion protein.

The prion glycoprotein (PrPC) is highly expressed in the nervous system as well as in other organs. Its functional roles in behavior have been examined mainly in non co-isogenic, wild-type and PrPC-deficient mice, which showed both age- and genotype-dependent differences. In general, however, effects of genetic background upon behavioral tests are mostly unclear when applied to aging rodents. The present study aimed to determine the effect of deletion of the prion protein on behavior of isogenic mice across different ages. We disclosed a genotype-dependent behavioral dissociation between either motor or cognitive tests, as a function of both age and test type. Remarkably, we also detected a clear age- and genotype-dependent difference in the variability of performance in a cognitive test. The current findings are relevant for both the interpretation of PrPC-related behavior, as well as for issues of reproducibility in studies of rodent behavior.

Antileishmanial Chemotherapy through Clemastine Fumarate Mediated Inhibition of the Leishmania Inositol Phosphorylceramide Synthase.

Current chemotherapeutics for leishmaniasis have multiple deficiencies, and there is a need for new safe, efficacious, and affordable medicines. This study describes a successful drug repurposing approach that identifies the over-the-counter antihistamine, clemastine fumarate, as a potential antileishmanial drug candidate. The screening for inhibitors of the sphingolipid synthase (inositol phosphorylceramide synthase, IPCS) afforded, following secondary screening against Leishmania major (Lmj) promastigotes, 16 active compounds. Further refinement through the dose response against LmjIPCS and intramacrophage L. major amastigotes identified clemastine fumarate with good activity and selectivity with respect to the host macrophage. On target engagement was supported by diminished sensitivity in a sphingolipid-deficient L. major mutant (ΔLmjLCB2) and altered phospholipid and sphingolipid profiles upon treatment with clemastine fumarate. The drug also induced an enhanced host cell response to infection indicative of polypharmacology. The activity was sustained across a panel of Old and New World Leishmania species, displaying an in vivo activity equivalent to the currently used drug, glucantime, in a mouse model of L. amazonensis infection. Overall, these data validate IPCS as an antileishmanial drug target and indicate that clemastine fumarate is a candidate for repurposing for the treatment of leishmaniasis.

Late Cognitive Consequences of Gestational Diabetes to the Offspring, in a New Mouse Model.

Gestational diabetes mellitus (GD) is a form of insulin resistance triggered during gestation, which affects approximately 10% of pregnant women. Although previously considered a transient condition with few long-term consequences, growing evidence suggest that GD may be linked to permanent metabolic and neurologic changes in the offspring. Currently available GD models fail to recapitulate the full spectrum of this disease, thus providing limited information about the true burden of this condition. Here, we describe a new mouse model of GD, based on the administration of an insulin receptor antagonist (S961, 30 nmol/kg s.c. daily) during pregnancy. Pregnant mice developed increased fasting glycemia and glucose intolerance in the absence of maternal obesity, with a return to normoglycemia shortly after parturition. Moreover, we showed that the adult offspring of GD dams presented pronounced metabolic and cognitive dysfunction when exposed to short-term high-fat diet (HFD). Our data demonstrate that S961 administration to pregnant mice comprises a valuable approach to study the complex pathophysiology of GD, as well as strategies focused on prevention and treatment of both the mother and the offspring. Our findings suggest that the offspring of GD mothers are more susceptible to metabolic and cognitive impairments when exposed to high-fat diet later in life, thus indicating that approaches to prevent and treat these late effects should be pursued.