Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Distinct Signaling Pathways for Autophagy-Driven Cell Death and Survival in Adult Hippocampal Neural Stem Cells.

Autophagy is a cellular catabolic process that degrades and recycles cellular materials. Autophagy is considered to be beneficial to the cell and organism by preventing the accumulation of toxic protein aggregates, removing damaged organelles, and providing bioenergetic substrates that are necessary for survival. However, autophagy can also cause cell death depending on cellular contexts. Yet, little is known about the signaling pathways that differentially regulate the opposite outcomes of autophagy. We have previously reported that insulin withdrawal (IW) or corticosterone (CORT) induces autophagic cell death (ACD) in adult hippocampal neural stem (HCN) cells. On the other hand, metabolic stresses caused by 2-deoxy-D-glucose (2DG) and glucose-low (GL) induce autophagy without death in HCN cells. Rather, we found that 2DG-induced autophagy was cytoprotective. By comparing IW and CORT conditions with 2DG treatment, we revealed that ERK and JNK are involved with 2DG-induced protective autophagy, whereas GSK-3ß regulates death-inducing autophagy. These data suggest that cell death and survival-promoting autophagy undergo differential regulation with distinct signaling pathways in HCN cells.

Fas-apoptotic inhibitory molecule 2 localizes to the lysosome and facilitates autophagosome-lysosome fusion through the LC3 interaction region motif-dependent interaction with LC3.

Fas-apoptotic inhibitory molecule 2 (FAIM2) is a member of the transmembrane BAX inhibitor motif-containing (TMBIM) family. TMBIM family is comprised of six anti-apoptotic proteins that suppress cell death by regulating endoplasmic reticulum Ca2+ homeostasis. Recent studies have implicated two TMBIM proteins, GRINA and BAX Inhibitor-1, in mediating cytoprotection via autophagy. However, whether FAIM2 plays a role in autophagy has been unknown. Here we show that FAIM2 localizes to the lysosomes at basal state and facilitates autophagy through interaction with microtubule-associated protein 1 light chain 3 proteins in human neuroblastoma SH-SY5Y cells. FAIM2 overexpression increased autophagy flux, while autophagy flux was impaired in shRNA-mediated knockdown (shFAIM2) cells, and the impairment was more evident in the presence of rapamycin. In shFAIM2 cells, autophagosome maturation through fusion with lysosomes was impaired, leading to accumulation of autophagosomes. A functional LC3-interacting region motif within FAIM2 was essential for the interaction with LC3 and rescue of autophagy flux in shFAIM2 cells while LC3-binding property of FAIM2 was dispensable for the anti-apoptotic function in response to Fas receptor-mediated apoptosis. Suppression of autophagosome maturation was also observed in a null mutant of Caenorhabditis elegans lacking xbx-6, the ortholog of FAIM2. Our study suggests that FAIM2 is a novel regulator of autophagy mediating autophagosome maturation through the interaction with LC3.

Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.

Distinct amyloid precursor protein processing machineries of the olfactory system.

Processing of amyloid precursor protein (APP) occurs through sequential cleavages first by ß-secretase and then by the γ-secretase complex. However, abnormal processing of APP leads to excessive production of ß-amyloid (Aß) in the central nervous system (CNS), an event which is regarded as a primary cause of Alzheimer's disease (AD). In particular, gene mutations of the γ-secretase complex-which contains presenilin 1 or 2 as the catalytic core-could trigger marked Aß accumulation. Olfactory dysfunction usually occurs before the onset of typical AD-related symptoms (eg, memory loss or muscle retardation), suggesting that the olfactory system may be one of the most vulnerable regions to AD. To date however, little is known about why the olfactory system is affected so early by AD prior to other regions. Thus, we examined the distribution of secretases and levels of APP processing in the olfactory system under either healthy or pathological conditions. Here, we show that the olfactory system has distinct APP processing machineries. In particular, we identified higher expressions levels and activity of γ-secretase in the olfactory epithelium (OE) than other regions of the brain. Moreover, APP c-terminal fragments (CTF) are markedly detected. During AD progression, we note increased expression of presenilin2 of γ-secretases in the OE, not in the OB, and show that neurotoxic Aß*56 accumulates more quickly in the OE. Taken together, these results suggest that the olfactory system has distinct APP processing machineries under healthy and pathological conditions. This finding may provide a crucial understanding of the unique APP-processing mechanisms in the olfactory system, and further highlights the correlation between olfactory deficits and AD symptoms.

A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflammasome activation.

Ro5-4864 and PK11195, prototypical synthetic ligands of translocator protein 18 kDa (TSPO), have shown anti-inflammatory effects in several models of inflammatory diseases; however, their biochemical mechanisms remain poorly understood. Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation as a part of the innate immune system, has been implicated in a variety of inflammatory diseases. Here, we demonstrate for the first time that TSPO ligands, especially Ro5-4864, potently suppressed ATP-induced NLRP3 inflammasome activation in THP-1 and BMDM cells. Detailed action mechanism was further investigated in THP-1 cells. Ro5-4864 efficiently attenuated NLRP3 translocation to mitochondria, inflammasome assembly/oligomerization, activation of caspase-1, and subsequent secretion of the mature forms of interleukin-1ß and -18. Ro5-4864 also reduced the production of mitochondrial superoxide and preserved the mitochondrial membrane potential in ATP-treated cells, suggesting that Ro5-4864 may act on mitochondria or more upstream targets in NLRP3 inflammasome signaling. We also observed the distinct effects of the TSPO ligands between THP-1 monocytes and macrophages, which suggested different NLRP3 inflammasome signaling depending on cell type. Collectively, our novel findings demonstrate that Ro5-4864 effectively inhibited ATP-induced NLRP3 inflammasome activation through the prevention of mitochondrial perturbation. Our results indicate Ro5-4864 as a promising candidate for the treatment of NLRP3 inflammasome-related diseases.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death.

Parkin-mediated responses against infection and wound involve TSPO-VDAC complex in Drosophila.

Parkin, an E3 ubuquitin ligase associated with Parkinson's disease (PD), has recently been implicated in mediating innate immunity. However, molecular details regarding parkin-mediated immune response remain to be elucidated. Here, we identified mitochondrial TSPO-VDAC complex to genetically interact with parkin in mediating responses against infection and wound in Drosophila. The loss-of-function mutation in parkin results in defective immune response against bacterial infection. Additionally, parkin mutant larvae showed hypersensitivity against wound regardless of bacterial infection. Interestingly, the combinatorial trans-heterozygotic mutations in parkin and TSPO, or parkin and VDAC showed similar lethal tendency with parkin homozygous mutants. Furthermore, knockdown of TSPO alone also resulted in defective responses to infection and wound analogously to parkin mutants. Taken together, we propose that parkin cooperates with TSPO-VDAC complex to mediate responses against infection and wound.

Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage.

BACKGROUND AND PURPOSE: Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage. METHODS: We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures. RESULTS: Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons. CONCLUSIONS: Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.

Imiquimod induces a Toll-like receptor 7-independent increase in intracellular calcium via IP(3) receptor activation.

Imiquimod is an itch-promoting, small, synthetic compound that is generally used to treat genital warts and basal cell carcinoma. The pruritogenic effect of imiquimod is considered to be due to TLR7 activation; however that idea has been challenged by our studies showing intact pruritogenic effects of imiquimod in TLR7 KO mice. Thus, the signaling pathways of imiquimod have not been completely elucidated. Here we investigated the novel effects of imiquimod on intracellular calcium ([Ca(2+)]i) signaling. We found that imiquimod induces [Ca(2+)]i increases in PC12 and F11 cells, and even in NIH-3T3 and HEK293T cells, which do not express TLR7. This [Ca(2+)]i increase was due to Ca(2+) release from the internal store without extracellular Ca(2+) influx. Neither FCCP, a mitochondrial Ca(2+) reuptake inhibitor, nor dantrolene, a ryanodine receptor inhibitor, affected the imiquimod-induced [Ca(2+)]i increase. However, 2APB, an IP3 receptor blocker, inhibited the imiquimod-induced [Ca(2+)]i increase. U73122, a PLCß inhibitor, failed to block the imiquimod-induced [Ca(2+)]i increase. These data indicate that imiquimod triggers IP3 receptor-dependent Ca(2+) signaling independently of TLR7.

Chronic unpredictable stress induces autophagic death of adult hippocampal neural stem cells.

Chronic psychological stress is a critical factor for neurological complications like anxiety disorders, dementia, and depression. Our previous results show that chronic restraint stress causes cognitive deficits and mood dysregulation by inducing autophagic death of adult hippocampal neural stem cells (NSCs). However, it is unknown whether other models of psychological stress also induce autophagic death of adult hippocampal NSCs. Here, we show that chronic unpredictable stress (CUS) for 10 days impaired memory function and increased anxiety in mice. Immunohistochemical staining with SOX2 and KI67 revealed a significant reduction in the number of NSCs in the hippocampus following exposure to CUS. However, these deficits were prevented by NSC-specific, inducible conditional deletion of Atg7. These findings suggest that autophagic death of adult hippocampal NSCs is a critical pathogenic mechanism underlying stress-induced brain disorders.

Role of the circadian nuclear receptor REV-ERBα in dorsal raphe serotonin synthesis in mood regulation.

Affective disorders are frequently associated with disrupted circadian rhythms. The existence of rhythmic secretion of central serotonin (5-hydroxytryptamine, 5-HT) pattern has been reported; however, the functional mechanism underlying the circadian control of 5-HTergic mood regulation remains largely unknown. Here, we investigate the role of the circadian nuclear receptor REV-ERBα in regulating tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme of 5-HT synthesis. We demonstrate that the REV-ERBα expressed in dorsal raphe (DR) 5-HTergic neurons functionally competes with PET-1-a nuclear activator crucial for 5-HTergic neuron development. In mice, genetic ablation of DR 5-HTergic REV-ERBα increases Tph2 expression, leading to elevated DR 5-HT levels and reduced depression-like behaviors at dusk. Further, pharmacological manipulation of the mice DR REV-ERBα activity increases DR 5-HT levels and affects despair-related behaviors. Our findings provide valuable insights into the molecular and cellular link between the circadian rhythm and the mood-controlling DR 5-HTergic systems.

Presenilin 2 N141I Mutation Induces Hyperimmunity by Immune Cell-specific Suppression of REV-ERBα without Altering Central Circadian Rhythm.

Circadian rhythm is a 24-hour cycle of behavioral and physiological changes. Disrupted sleep-wake patterns and circadian dysfunction are common in patients of Alzheimer Disease (AD) and are closely related with neuroinflammation. However, it is not well known how circadian rhythm of immune cells is altered during the progress of AD. Previously, we found presenilin 2 (Psen2) N141I mutation, one of familial AD (FAD) risk genes, induces hyperimmunity through the epigenetic repression of REV-ERBα expression in microglia and bone marrow-derived macrophage (BMDM) cells. Here, we investigated whether repression of REV-ERBα is associated with dysfunction of immune cell-endogenous or central circadian rhythm by analyses of clock genes expression and cytokine secretion, bioluminescence recording of rhythmic PER2::LUC expression, and monitoring of animal behavioral rhythm. Psen2 N141I mutation down-regulated REV-ERBα and induced selective over-production of IL-6 (a well-known clock-dependent cytokine) following the treatment of toll-like receptor (TLR) ligands in microglia, astrocytes, and BMDM. Psen2 N141I mutation also lowered amplitude of intrinsic daily oscillation in these immune cells representatives of brain and periphery. Of interest, however, the period of daily rhythm remained intact in immune cells. Furthermore, analyses of the central clock and animal behavioral rhythms revealed that central clock remained normal without down-regulation of REV-ERBα. These results suggest that Psen2 N141I mutation induces hyperimmunity mainly through the suppression of REV-ERBα in immune cells, which have lowered amplitude but normal period of rhythmic oscillation. Furthermore, our data reveal that central circadian clock is not affected by Psen2 N141I mutation.

Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα.

Hyperimmunity drives the development of Alzheimer disease (AD). The immune system is under the circadian control, and circadian abnormalities aggravate AD progress. Here, we investigate how an AD-linked mutation deregulates expression of circadian genes and induces cognitive decline using the knock-in (KI) mice heterozygous for presenilin 2 N141I mutation. This mutation causes selective overproduction of clock gene-controlled cytokines through the DNA hypermethylation-mediated repression of REV-ERBα in innate immune cells. The KI/+ mice are vulnerable to otherwise innocuous, mild immune challenges. The antipsychotic chlorpromazine restores the REV-ERBα level by normalizing DNA methylation through the inhibition of PI3K/AKT1 pathway, and prevents the overexcitation of innate immune cells and cognitive decline in KI/+ mice. These results highlight a pathogenic link between this AD mutation and immune cell overactivation through the epigenetic suppression of REV-ERBα.

Aß-induced mitochondrial dysfunction in neural progenitors controls KDM5A to influence neuronal differentiation.

Mitochondria in neural progenitors play a crucial role in adult hippocampal neurogenesis by being involved in fate decisions for differentiation. However, the molecular mechanisms by which mitochondria are related to the genetic regulation of neuronal differentiation in neural progenitors are poorly understood. Here, we show that mitochondrial dysfunction induced by amyloid-beta (Aß) in neural progenitors inhibits neuronal differentiation but has no effect on the neural progenitor stage. In line with the phenotypes shown in Alzheimer's disease (AD) model mice, Aß-induced mitochondrial damage in neural progenitors results in deficits in adult hippocampal neurogenesis and cognitive function. Based on hippocampal proteome changes after mitochondrial damage in neural progenitors identified through proteomic analysis, we found that lysine demethylase 5A (KDM5A) in neural progenitors epigenetically suppresses differentiation in response to mitochondrial damage. Mitochondrial damage characteristically causes KDM5A degradation in neural progenitors. Since KDM5A also binds to and activates neuronal genes involved in the early stage of differentiation, functional inhibition of KDM5A consequently inhibits adult hippocampal neurogenesis. We suggest that mitochondria in neural progenitors serve as the checkpoint for neuronal differentiation via KDM5A. Our findings not only reveal a cell-type-specific role of mitochondria but also suggest a new role of KDM5A in neural progenitors as a mediator of retrograde signaling from mitochondria to the nucleus, reflecting the mitochondrial status.

Activation of autophagy through modulation of 5'-AMP-activated protein kinase protects pancreatic beta-cells from high glucose.

Chronic hyperglycaemia is detrimental to pancreatic beta-cells by causing impaired insulin secretion and diminished beta-cell function through glucotoxicity. Understanding the mechanisms underlying beta-cell survival is crucial for the prevention of beta-cell failure associated with glucotoxicity. Autophagy is a dynamic lysosomal degradation process that protects organisms against metabolic stress. To date, little is known about the physiological function of autophagy in the pathogenesis of diabetes. In the present study, we explored the roles of autophagy in the survival of pancreatic beta-cells exposed to high glucose using pharmacological and genetic manipulation of autophagy. We demonstrated that chronic high glucose increases autophagy in rat INS-1 (832/13) cells and pancreatic islets, and that this increase is enhanced by inhibition of 5'-AMP-activated protein kinase. Our results also indicate that stimulation of autophagy rescues pancreatic beta-cells from high-glucose-induced cell death and inhibition of autophagy augments caspase-3 activation, suggesting that autophagy plays a protective role in the survival of pancreatic beta-cells. Greater knowledge of the molecular mechanisms linking autophagy and beta-cell survival may unveil novel therapeutic targets needed to preserve beta-cell function.

GSK3B induces autophagy by phosphorylating ULK1.

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis.

Autophagy as a decisive process for cell death.

Autophagy is an intracellular catabolic pathway in which cellular constituents are engulfed by autophagosomes and degraded upon autophagosome fusion with lysosomes. Autophagy serves as a major cytoprotective process by maintaining cellular homeostasis and recycling cytoplasmic contents. However, emerging evidence suggests that autophagy is a primary mechanism of cell death (autophagic cell death, ACD) and implicates ACD in several aspects of mammalian physiology, including tumor suppression and psychological disorders. However, little is known about the physiological roles and molecular mechanisms of ACD. In this review, we document examples of ACD and discuss recent progress in our understanding of its molecular mechanisms.

Cx3cr1CreERT2-driven Atg7 deletion in adult mice induces intestinal adhesion.

Microglia are macrophages resident in the central nervous system. C-X3-C motif chemokine receptor 1 (CX3CR1) is a Gαi-coupled seven-transmembrane protein exclusively expressed in the mononuclear phagocyte system including microglia, as well as intestinal and kidney macrophages. Cx3cr1CreERT2 mice express Cre recombinase in a tamoxifen-inducible manner and have been widely used to delete target genes in microglia, since microglia are long-lived cells and outlive peripheral macrophages, which continuously turn over and lose their gene modification over time. ATG7 is an E1-like enzyme that plays an essential role in two ubiquitin-like reactions, ATG12-ATG5 conjugation and LC3-lipidation in autophagy. To study the role of ATG7 in adult microglia, we generated Cx3cr1CreERT2:Atg7fl/fl mice and deleted Atg7 at the age of 8 weeks, and found induction of intestinal adhesion. Since intestinal adhesion is caused by excessive inflammation, these results suggest that deletion of Atg7 in intestinal macrophages even for a short time results in inflammation that cannot be rescued by replenishment with wild-type intestinal macrophages. Our finding suggests that, depending on the roles of the gene, Cx3cr1-Cre-mediated gene deletion may yield unanticipated physiological outcomes outside the central nervous system, and careful necropsy is necessary to assure the microglia-specific roles of the target gene.

Translocator protein (TSPO): the new story of the old protein in neuroinflammation.

Translocator protein (TSPO), also known as peripheral benzodiazepine receptor, is a transmembrane protein located on the outer mitochondria membrane (OMM) and mainly expressed in glial cells in the brain. Because of the close correlation of its expression level with neuropathology and therapeutic efficacies of several TSPO binding ligands under many neurological conditions, TSPO has been regarded as both biomarker and therapeutic target, and the biological functions of TSPO have been a major research focus. However, recent genetic studies with animal and cellular models revealed unexpected results contrary to the anticipated biological importance of TSPO and cast doubt on the action modes of the TSPO-binding drugs. In this review, we summarize recent controversial findings on the discrepancy between pharmacological and genetic studies of TSPO and suggest some future direction to understand this old and mysterious protein. [BMB Reports 2020; 53(1): 20-27].