Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release in the ventral midbrain.

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment through a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity and shows a calcium sensitivity that is comparable to that of axonal release. We find that the molecular mechanism of STD DA release differs from axonal release with regard to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knockout of Syt4 or Syt7 is not sufficient to reduce STD DA release, the removal of both isoforms reduces this release by approximately 50%, leaving axonal release unimpaired. Our work unveils clear differences in the mechanisms of STD and axonal DA release.

Circular versus linear RNA topology: different modes of RNA-RNA interactions in vitro and in human cells.

Circular RNA is progressively reported to occur in various species including mammals where it is thought to be involved in the post-transcriptional regulation of gene expression, partly via interactions with microRNA. Here, we asked whether the circular topology causes functional differences to linear forms when interacting with short RNA strands in vitro and in human cells. Kinetic studies with human bladder cancer-derived synthetic circular RNA versus linear transcripts, respectively, with short oligoribonucleotides showed similar association rates for both topologies. Conversely, a substantial topology-related difference was measured for the activation entropy and the activation enthalpy of RNA-RNA annealing. This finding strongly indicates a significant difference of the mechanism of RNA-RNA interactions. To investigate whether these characteristics of circular RNA are biologically meaningful we performed transient transfection experiments with a microRNA-regulated expression system for luciferase in bladder cancer-derived cells. We co-transfected linear or circular RNA containing one microRNA binding site for the target-suppressing microRNA mlet7a. Here, the circular isoform showed a strongly increased competition with microRNA function versus linear versions. In summary, this study suggests novel topology-related characteristics of RNA-RNA interactions involving circRNA in vitro and in living cells.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

Impaired endothelium-mediated cerebrovascular reactivity promotes anxiety and respiration disorders in mice.

Carbon dioxide (CO2), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H+ receptor, and endothelial Gαq/11 proteins mediate the CO2/H+ effect on cerebrovascular reactivity in mice. CO2/H+ leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO2, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO2 dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO2 effect on anxiety. Even at atmospheric CO2 concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO2 is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.