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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.

Nitric oxide (NO) has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved -specially the guanylyl cyclase-independent ones -has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that NO induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase C gamma 1 (PLC gamma 1) to this same tyrosine residue. Addi-tionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP-2 and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it to-wards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the visual cortex, and promoted a shift in ocular dominance upon monocular deprivation -an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.

COVID-19 is a disease caused by coronavirus SARS-CoV-2. In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms that can last from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to infection of brain cells. We found that a small fraction of cortical 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), which was expressed at very low levels. Although only a limited number of neurons was infectable, the infection was productive, as demonstrated by the presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout the neuronal cell, and an increase in viral RNA in the culture medium within the first 48 h of infection (viral release). The productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface protein spike, which can occur at the plasma membrane or from endo/lysosomes. Using specific combinations of small-molecule inhibitors, we found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases found on the cell surface, including TMPRSS2. In contrast, the infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K) that regulates endosomal maturation.ImportanceCOVID-19 is a disease caused by 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. Whether these symptoms are linked to brain infection is not clear. The mechanism of the virus entry into neurons has also not been characterized. Here we investigated SARS-CoV-2 infection using a human iPSC-derived neural cell model and found that a small fraction of cortical neurons was naturally susceptible to infection. The infection depended on the ACE2 receptor and was productive. We also found that the virus used the late endosomal/lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of the cellular phosphatidyl-inositol 5 kinase.

Nitric oxide has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that nitric oxide induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase Cγ1 (PLCγ1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP2M and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the primary visual cortex, and promoted a shift in ocular dominance upon monocular deprivation in the visual cortex - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.Significance statementWe described the nitration of TRKB receptors at the tyrosine residue 816 as a new post-translational modification (PTM) that restrains the signaling of the neurotrophic factor BDNF in neurons. This new PTM leads to endocytosis and degradation of the TRKB receptors. Intriguingly, this mechanism is tonically active under physiological conditions in vivo, and it is important for restricting ocular dominance plasticity in the visual cortex. This mechanism directly links two major systems involved in brain plasticity, BDNF/TRKB and nitric oxide. Our data provides a model for how NO production from nNOS can compromise TRKB function, and for the effects of nNOS inhibitors promoting plasticity.

Perineuronal nets (PNNs) are an extracellular matrix structure rich in chondroitin sulfate proteoglycans (CSPGs), which preferentially encase parvalbumin-containing (PV+) interneurons. PNNs restrict cortical network plasticity but the molecular mechanisms involved are unclear. We found that reactivation of ocular dominance plasticity in the adult visual cortex induced by chondroitinase ABC (chABC)-mediated PNN removal requires intact signaling by the neurotrophin receptor TRKB in PV+neurons. Additionally, we demonstrate that chABC increases TRKB phosphorylation (pTRKB), while PNN component aggrecan attenuates brain-derived neurotrophic factor (BDNF)-induced pTRKB in cortical neurons in culture. We further found that protein tyrosine phosphatase σ (PTPσ, PTPRS), receptor for CSPGs, interacts with TRKB and restricts TRKB phosphorylation. PTPσ deletion increases phosphorylation of TRKBin vitroandin vivoin male and female mice, and juvenile-like plasticity is retained in the visual cortex of adult PTPσ-deficient mice (PTPσ+/−). The antidepressant drug fluoxetine, which is known to promote TRKB phosphorylation and reopen critical period-like plasticity in the adult brain, disrupts the interaction between TRKB and PTPσ by binding to the transmembrane domain of TRKB. We propose that both chABC and fluoxetine reopen critical period-like plasticity in the adult visual cortex by promoting TRKB signaling in PV+neurons through inhibition of TRKB dephosphorylation by the PTPσ-CSPG complex.SIGNIFICANCE STATEMENTCritical period-like plasticity can be reactivated in the adult visual cortex through disruption of perineuronal nets (PNNs) by chondroitinase treatment, or by chronic antidepressant treatment. We now show that the effects of both chondroitinase and fluoxetine are mediated by the neurotrophin receptor TRKB in parvalbumin-containing (PV+) interneurons. We found that chondroitinase-induced visual cortical plasticity is dependent on TRKB in PV+neurons. Protein tyrosine phosphatase σ (PTPσ, PTPRS), a receptor for PNNs, interacts with TRKB and inhibits its phosphorylation, and chondroitinase treatment or deletion of PTPσ increases TRKB phosphorylation. Antidepressant fluoxetine disrupts the interaction between TRKB and PTPσ, thereby increasing TRKB phosphorylation. Thus, juvenile-like plasticity induced by both chondroitinase and antidepressant treatment is mediated by TRKB activation in PV+interneurons.

Therapeutical approaches providing effective medication for Alzheimer’s disease (AD) patients after disease onset are urgently needed. Repurposing FDA approved drugs like fingolimod (FTY720) for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms. Here, we addressed whether the FDA approved drug fingolimod rescues AD related synaptic deficits and memory dysfunction in an APP/PS1 AD mouse model when medication starts after onset of symptoms (at 5 months). Male mice received intraperitoneal injections of fingolimod for 1-2 months starting at 5-6 months. This treatment rescued spine density as well as long-term potentiation in hippocampal CA1 pyramidal neurons, and ameliorated dysfunctional hippocampus-dependent memory that was observed in untreated APP/PS1 animals at 6-7 months of age. Immunohistochemical analysis with markers of microgliosis (Iba1) and astrogliosis (GFAP) revealed that our fingolimod treatment regime strongly down regulated neuro-inflammation in the hippocampus and cortex of this AD model. These effects were accompanied by a moderate reduction of Aβ accumulation in hippocampus and cortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology and related memory performance deficits observed in untreated AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes.

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANG II). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety‐ and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the later. Our data suggested an anxiogenic‐ and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.