Regulation of autophagic cell death by glycogen synthase kinase-3 beta in adult hippocampal neural stem cells following insulin withdrawal

Background: Neural stem cells (NSCs) hold great potential for the treatment of neurodegenerative diseases. However, programmed cell death (PCD) provoked by the harsh conditions evident in the diseased brain greatly undermines the potential of NSCs. Currently, the mechanisms of PCD that effect NSCs remain largely unknown. Results: We have previously reported that hippocampal neural stem (HCN) cells derived from the adult rat brain undergo autopahgic cell death (ACD) following insulin withdrawal without hallmarks of apoptosis despite their normal apoptotic capabilities. In this study, we demonstrate that glycogen synthase kinase 3β (GSK-3β) induces ACD in insulin-deprived HCN cells. Both pharmacological and genetic inactivation of GSK-3β significantly decreased ACD, while activation of GSK-3β increased autophagic flux and caused more cell death without inducing apoptosis following insulin withdrawal. In contrast, knockdown of GSK-3α barely affected ACD, lending further support to the critical role of GSK-3β. Conclusion: Collectively, these data demonstrate that GSK-3β is a key regulator of ACD in HCN cells following insulin withdrawal. The absence of apoptotic indices in GSK-3β-induced cell death in insulin-deprived HCN cells corroborates the notion that HCN cell death following insulin withdrawal represents the genuine model of ACD in apoptosis-intact mammalian cells and identifies GSK-3β as a key negative effector of NSC survival downstream of insulin signaling. © 2015 Ha et al.; licensee BioMed Central.1

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 alpha 2 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.1

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. © 2021, The Author(s).1

성체해마신경줄기세포의 세포예정사멸과 성상세포분화 과정에서 자가포식현상의 역할 규명

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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-3β regulates death-inducing autophagy. These data suggest that cell death and survival-promoting autophagy undergo differential regulation with distinct signaling pathways in HCN cells. © 2023 by the authors.TRU

TLR4 (toll-like receptor 4) activation suppresses autophagy through inhibition of FOXO3 and impairs phagocytic capacity of microglia

Macroautophagy/autophagy is a lysosome-dependent catabolic process for the turnover of proteins and organelles in eukaryotes. Autophagy plays an important role in immunity and inflammation, as well as metabolism and cell survival. Diverse immune and inflammatory signals induce autophagy in macrophages through pattern recognition receptors, such as toll-like receptors (TLRs). However, the physiological role of autophagy and its signaling mechanisms in microglia remain poorly understood. Microglia are phagocytic immune cells that are resident in the central nervous system and share many characteristics with macrophages. Here, we show that autophagic flux and expression of autophagy-related (Atg) genes in microglia are significantly suppressed upon TLR4 activation by lipopolysaccharide (LPS), in contrast to their stimulation by LPS in macrophages. Metabolomics analysis of the levels of phosphatidylinositol (PtdIns) and its 3-phosphorylated form, PtdIns3P, in combination with bioinformatics prediction, revealed an LPS-induced reduction in the synthesis of PtdIns and PtdIns3P in microglia but not macrophages. Interestingly, inhibition of PI3K, but not MTOR or MAPK1/3, restored autophagic flux with concomitant dephosphorylation and nuclear translocation of FOXO3. A constitutively active form of FOXO3 also induced autophagy, suggesting FOXO3 as a downstream target of the PI3K pathway for autophagy inhibition. LPS treatment impaired phagocytic capacity of microglia, including MAP1LC3B/LC3-associated phagocytosis (LAP) and amyloid β (Aβ) clearance. PI3K inhibition restored LAP and degradation capacity of microglia against Aβ. These findings suggest a unique mechanism for the regulation of microglial autophagy and point to the PI3K-FOXO3 pathway as a potential therapeutic target to regulate microglial function in brain disorders. Abbreviations: Atg: autophagy-related gene; Aβ: amyloid-β; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; BMDM: bone marrow-derived macrophage; CA: constitutively active; CNS: central nervous system; ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1; FOXO: forkhead box O; ELISA:enzyme-linked immunosorbent assay; HBSS: Hanks balanced salt solution; LAP: LC3-associated phagocytosis; MAP1LC3B: microtubule-associated protein 1 light chain 3; LPS: lipopolysaccharide; LY: LY294002; MTOR: mechanistic target of rapamycin kinase; Pam3CSK4: N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PLA: proximity ligation assay; Poly(I:C): polyinosinic-polycytidylic acid; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; TLR: Toll-like receptor; TNF: tumor necrosis factor; TFEB: transcription factor EB; TSPO: translocator protein. © 2018 Informa UK Limited, trading as Taylor & Francis Group.1

Quantitative mass spectrometric analysis of the mouse cerebral cortex after ischemic stroke.

Ischemic strokes result in the death of brain tissue and a wave of downstream effects, often leading to lifelong disabilities or death. However, the underlying mechanisms of ischemic damage and repair systems remain largely unknown. In order to better understand these mechanisms, TMT-isobaric mass tagging and mass spectrometry were conducted on brain cortex extracts from mice subjected to one hour of middle cerebral artery occlusion (MCAO) and after one hour of reperfusion. In total, 2,690 proteins were identified and quantified, out of which 65% of the top 5% of up- and down-regulated proteins were found to be significant (p < 0.05). Network-based gene ontology analysis was then utilized to cluster all identified proteins by protein functional groups and cellular roles. Although three different cellular functions were identified-organelle outer membrane proteins, cytosolic ribosome proteins, and spliceosome complex proteins-several functional domains were found to be common. Of these, organelle outer membrane proteins were downregulated whereas cytosolic ribosome and spliceosome complex proteins were upregulated, indicating that major molecular events post-stroke were translation-associated and subsequent signaling pathways (e.g., poly (ADP-ribose) (PAR) dependent cell death). By approaching stroke analyses via TMT-isobaric mass tagging, the work herein presents a grand scope of protein-based molecular mechanisms involved with ischemic stroke recovery

Efficacy and Safety of <i>Lactobacillus Plantarum</i> C29-Fermented Soybean (DW2009) in Individuals with Mild Cognitive Impairment: A 12-Week, Multi-Center, Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Early intervention using dietary supplements may be effective in alleviating cognitive impairment among individuals with mild cognitive impairment (MCI). This study investigated the efficacy and safety of Lactobacillus plantarum C29-fermented soybean (DW2009) as a nutritional supplement for cognitive enhancement. One hundred individuals with MCI were randomly assigned to take DW2009 (800 mg/day, n = 50) or placebo (800 mg/day, n = 50) for 12 weeks. The primary outcome measure was change in the composite score of cognitive functions related to memory and attention, measured by computerized neurocognitive function tests. Associations between changes in serum brain-derived neurotrophic factor (BDNF) levels and cognitive performance for each treatment group were evaluated. Compared to the placebo group, the DW2009 group showed greater improvements in the combined cognitive functions (z = 2.36, p for interaction = 0.02), especially in the attention domain (z = 2.34, p for interaction = 0.02). Cognitive improvement was associated with increased serum BDNF levels after consumption of DW2009 (t = 2.83, p = 0.007). The results of this clinical trial suggest that DW2009 can be safely administered to enhance cognitive function in individuals with MCI. Increased serum BDNF levels after administering DW2009 may provide preliminary insight into the underlying effects of cognitive improvement, which suggests the importance of the gut-brain axis in ameliorating cognitive deficits in MCI

Magnetically actuated microrobots as a platform for stem cell transplantation

Magnetic microrobots were developed for three-dimensional culture and the precise delivery of stem cells in vitro, ex vivo, and in vivo. Hippocampal neural stem cells attached to the microrobots proliferated and differentiated into astrocytes, oligodendrocytes, and neurons. Moreover, microrobots were used to transport colorectal carcinoma cancer cells to tumor microtissue in a body-on-a-chip, which comprised an in vitro liver-tumor microorgan network. The microrobots were also controlled in a mouse brain slice and rat brain blood vessel. Last, microrobots carrying mesenchymal stem cells derived from human nose were manipulated inside the intraperitoneal cavity of a nude mouse. The results indicate the potential of microrobots for the culture and delivery of stem cells. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works1

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 Ca2+ 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 Ca2+ 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. © 2015 AlphaMed Press.