New perspectives on natural flavonoids on COVID-19-induced lung injuries.

The SARS-CoV-2 virus, responsible for COVID-19, spread rapidly worldwide and became a pandemic in 2020. In some patients, the virus remains in the respiratory tract, causing pneumonia, respiratory failure, acute respiratory distress syndrome (ARDS), and sepsis, leading to death. Natural flavonoids (aglycone and glycosides) possess broad biological activities encompassing antiinflammatory, antiviral, antitumoral, antiallergic, antiplatelet, and antioxidant effects. While many studies have focused on the effects of natural flavonoids in experimental models, reports based on clinical trials are still insufficient. In this review, we highlight the effects of flavonoids in controlling pulmonary diseases, particularly the acute respiratory distress syndrome, a consequence of COVID-19, and their potential use in coronavirus-related diseases. Furthermore, we also focus on establishing a relationship between biological potential and chemical aspects of related flavonoids and discuss several possible mechanisms of action, pointing out some possible effects on COVID-19.

α-Synuclein Overexpression Induces Lysosomal Dysfunction and Autophagy Impairment in Human Neuroblastoma SH-SY5Y.

Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.

Cannabidiol induces autophagy via ERK1/2 activation in neural cells.

Autophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

Glutamate induces autophagy via the two-pore channels in neural cells.

NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.

NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca(2+) release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. In the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca(2+) by TPCs. We show that Ca(2+) signals mediated by NAADP were inhibited by disrupting Ca(2+) handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca(2+) signals. Desensitizing the NAADP pathway inhibited Ca(2+)-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca(2+) release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells.

Effect of chronic sleep restriction and aging on calcium signaling and apoptosis in the hippocampus of young and aged animals.

Aging leads to progressive deterioration of physiological function and diminished responses to environmental stress. Organic and functional alterations are frequently observed in elderly subjects. Although chronic sleep loss is observed during senescence, little is known about the impact of insufficient sleep on cellular function in aging neurons. Disruption of neuronal calcium (Ca²âº) signaling is related to impaired neuronal function and cell death. It has been hypothesized that sleep deprivation may compromise neuronal stability and induce cell death in young neurons; however, it is necessary to evaluate the impact of aging on this process. Therefore, the aim of this study was to evaluate the effects of chronic sleep restriction (CSR) on Ca²âº signaling and cell death in the hippocampus of young and aged animals. We found that glutamate and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced a greater elevation in cytosolic Ca²âº ([Ca²âº](c)) in hippocampal slices from aged rats subjected to CSR compared to age-matched controls. Interestingly, aged-matched controls showed a reduced Ca²âº response to glutamate and FCCP, relative to both CSR and control young animals. Apoptotic nuclei were observed in aged rats from both treatment groups; however, the profile of apoptotic nuclei in aged CSR rats was highly variable. Bax and Bcl-2 protein expression did not change with aging in the CSR groups. Our study indicates that aging promotes changes in Ca²âº signaling, which may also be affected by CSR. These age-dependent changes in Ca²âº signaling may increase cellular vulnerability during CSR and contribute to Ca²âº signaling dysregulation, which may ultimately induce cell death.

Differential proliferative response of the ventral prostate and seminal vesicle to testosterone replacement.

We have investigated epithelial cell proliferation and the rate of glandular recovery of the ventral prostate (VP) and seminal vesicle (SV) promoted by testosterone replacement (TR) in castration-induced regressed glands. Adult male Wistar rats were castrated and, after 21 days, they were treated with testosterone propionate (4 mg/kg/day). Intact (CT) and castrated rats without TR (CS) were also analysed. VP and SV were processed for histochemistry, morphometric-stereological analysis and immunocytochemistry to determine the PCNA index (PI). After 10 days of TR, the VP weight reached approximately 72% of the CT values, while the SV weight exceeded approximately 17% of the CT values. By the third day of TR, VP and SV presented a mean PI of 34% and 94% for distal region and 14% and 22% for proximal region, respectively. SV also had more luminal cells PCNA-positive than VP, mainly in the distal region. The PI values fell on days 5, 7 and 10, but were still higher than CT. These findings indicate that epithelial cells from involuted SV are more responsive to TR than those from VP when stimulated to proliferate and replace the luminal cell population, suggesting a different mechanism regulating cell proliferation in response to androgenic stimuli.