SARS-CoV-2 Infected Pediatric Cerebral Cortical Neurons: Transcriptomic Analysis and Potential Role of Toll-like Receptors in Pathogenesis

Different mechanisms were proposed as responsible for COVID-19 neurological symptoms but a clear one has not been established yet. In this work we aimed to study SARS-CoV-2 capacity to infect pediatric human cortical neuronal HCN-2 cells, studying the changes in the transcriptomic profile by next generation sequencing. SARS-CoV-2 was able to replicate in HCN-2 cells, that did not express ACE2, confirmed also with Western blot, and TMPRSS2. Looking for pattern recognition receptor expression, we found the deregulation of scavenger receptors, such as SR-B1, and the downregulation of genes encoding for Nod-like receptors. On the other hand, TLR1, TLR4 and TLR6 encoding for Toll-like receptors (TLRs) were upregulated. We also found the upregulation of genes encoding for ERK, JNK, NF-κB and Caspase 8 in our transcriptomic analysis. Regarding the expression of known receptors for viral RNA, only RIG-1 showed an increased expression; downstream RIG-1, the genes encoding for TRAF3, IKKε and IRF3 were downregulated. We also found the upregulation of genes encoding for chemokines and accordingly we found an increase in cytokine/chemokine levels in the medium. According to our results, it is possible to speculate that additionally to ACE2 and TMPRSS2, also other receptors may interact with SARS-CoV-2 proteins and mediate its entry or pathogenesis in pediatric cortical neurons infected with SARS-CoV-2. In particular, TLRs signaling could be crucial for the neurological involvement related to SARS-CoV-2 infection.

Urokinase-type plasminogen activator promotes N-cadherin-mediated synaptic recovery in the ischemic brain

Urokinase-type plasminogen activator (uPA) is a serine proteinase that catalyzes the generation of plasmin on the cell surface and activates cell signaling pathways that promote remodeling and repair. Neuronal cadherin (NCAD) is a transmembrane protein that in the mature brain mediates the formation of synaptic contacts in the II/III and V cortical layers. Our studies show that uPA is preferentially found in the II/III and V cortical laminae of the gyrencephalic cortex of the non-human primate. Furthermore, we found that in murine cerebral cortical neurons and induced pluripotent stem cell (iPSC)-derived neurons prepared from healthy human donors, most of this uPA is associated with pre-synaptic vesicles. Our in vivo experiments revealed that in both, the gyrencephalic cortex of the non-human primate and the lissecephalic murine brain, cerebral ischemia decreases the number of intact synaptic contacts and the expression of uPA and NCAD in a band of tissue surrounding the necrotic core. Additionally, our in vitro data show that uPA induces the synthesis of NCAD in cerebral cortical neurons, and in line with these observations, intravenous treatment with recombinant uPA three hours after the onset of cerebral ischemia induces NCAD-mediated repair of synaptic contacts in the area surrounding the necrotic core.

Cannabinoids promote NREM sleep via inhibition of prefrontal cortico-hypothalamic projections

Cannabinoids promote non-rapid eye movement (NREM) sleep, but its underlying mechanism is not known. Here we find that cannabinoid promotion of NREM sleep is inhibited by cannabinoid antagonist injection systemically or into the dorsomedial hypothalamus (DMH), where cannabinoids selectively inhibit glutamatergic inputs synapsing with glutamatergic but not GABAergic neurons. Photoactivation of DMH-projecting ventromedial prefrontal cortical (vmPFC) neurons, their terminals, or their postsynaptic DMH neurons rapidly switches NREM sleep to wakefulness, which is blocked by photoinhibition of DMH outputs. Chemoactivation of DMH glutamatergic but not GABAergic neurons innervated by vmPFCs promotes wakefulness and suppresses NREM sleep, whereas chemoinhibition of vmPFC projections in DMHs produces opposite effects by mimicking cannabinoid effects. DMH-projecting vmPFC neurons are inhibited during NREM sleep and activated during wakefulness. Chemoactivation of DMH-projecting vmPFC neurons blocks cannabinoid promotion of NREM sleep and suppression of wakefulness. Thus, vmPFC neurons innervating DMHs represent the first identified set of cerebral cortical neurons for promotion of physiological wakefulness and suppression of NREM sleep, while cannabinoid inhibition of vmPFC projections in DMHs promotes NREM sleep and suppresses wakefulness.

Amyloid-beta peptide and phosphorylated tau in the frontopolar cerebral cortex and in the cerebellum of toothed whales: aging versus hypoxia

ABSTRACTHypoxia could be a possible risk factor for neurodegenerative alterations in cetaceans’ brain. Among toothed whales, the beaked whales are particularly cryptic and routinely dive deeper than 1000 m for about 1 h in order to hunt squids and fishes. Samples of frontal cerebral and cerebellar cortex were collected from nine animals, representing six different species of the suborder Odontoceti. Immunohistochemical analysis employed anti-β-amyloid (Aβ) and anti-neurofibrillary tangle (NFT) antibodies. Six of nine (67%) animals showed positive immunolabeling for Aβ and/or NFT. The most striking findings were intranuclear Aβ immunopositivity in cerebral cortical neurons and NFT immunopositivity in cerebellar Purkinje neurons with granulovacuolar degeneration. Aβ plaques were also observed in one elderly animal. Herein, we present immunohistopathological findings classic of Alzheimer's and other neurodegenerative diseases in humans. Our findings could be linked to hypoxic phenomena, as they were more extensive in beaked whales. Despite their adaptations, cetaceans could be vulnerable to sustained and repetitive brain hypoxia.