Cx3cr1(CreERT2)-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. Cx3cr1 CreERT2 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 Cx3cr1 CreERT2 :Atg7 fl/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. © 2020 The Author(s).1

Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/beta-catenin signaling in the hypothalamus

Background: Insulin plays diverse roles in the brain. Although insulin produced by pancreatic β-cells that crosses the blood-brain barrier is a major source of brain insulin, recent studies suggest that insulin is also produced locally within the brain. However, the mechanisms underlying the production of brain-derived insulin (BDI) are not yet known. Results: Here, we examined the effect of Wnt3a on BDI production in a hypothalamic cell line and hypothalamic tissue. In N39 hypothalamic cells, Wnt3a treatment significantly increased the expression of the Ins2 gene, which encodes the insulin isoform predominant in the mouse brain, by activating Wnt/β-catenin signaling. The concentration of insulin was higher in culture medium of Wnt3a-treated cells than in that of untreated cells. Interestingly, neurogenic differentiation 1 (NeuroD1), a target of Wnt/β-catenin signaling and one of transcription factors for insulin, was also induced by Wnt3a treatment in a time- and dose-dependent manner. In addition, the treatment of BIO, a GSK3 inhibitor, also increased the expression of Ins2 and NeuroD1. Knockdown of NeuroD1 by lentiviral shRNAs reduced the basal expression of Ins2 and suppressed Wnt3a-induced Ins2 expression. To confirm the Wnt3a-induced increase in Ins2 expression in vivo, Wnt3a was injected into the hypothalamus of mice. Wnt3a increased the expression of NeuroD1 and Ins2 in the hypothalamus in a manner similar to that observed in vitro. Conclusion: Taken together, these results suggest that BDI production is regulated by the Wnt/β-catenin/NeuroD1 pathway in the hypothalamus. Our findings will help to unravel the regulation of BDI production in the hypothalamus.1

Parkin Promotes Mitophagic Cell Death in Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal

Regulated cell death (RCD) plays a fundamental role in human health and disease. Apoptosis is the best-studied mode of RCD, but the importance of other modes has recently been gaining attention. We have previously demonstrated that adult rat hippocampal neural stem (HCN) cells undergo autophagy-dependent cell death (ADCD) following insulin withdrawal. Here, we show that Parkin mediates mitophagy and ADCD in insulin-deprived HCN cells. Insulin withdrawal increased the amount of depolarized mitochondria and their colocalization with autophagosomes. Insulin withdrawal also upregulated both mRNA and protein levels of Parkin, gene knockout of which prevented mitophagy and ADCD. c-Jun is a transcriptional repressor of Parkin and is degraded by the proteasome following insulin withdrawal. In insulin-deprived HCN cells, Parkin is required for Ca2+ accumulation and depolarization of mitochondria at the early stages of mitophagy as well as for recognition and removal of depolarized mitochondria at later stages. In contrast to the pro-death role of Parkin during mitophagy, Parkin deletion rendered HCN cells susceptible to apoptosis, revealing distinct roles of Parkin depending on different modes of RCD. Taken together, these results indicate that Parkin is required for the induction of ADCD accompanying mitochondrial dysfunction in HCN cells following insulin withdrawal. Since impaired insulin signaling is implicated in hippocampal deficits in various neurodegenerative diseases and psychological disorders, these findings may help to understand the mechanisms underlying death of neural stem cells and develop novel therapeutic strategies aiming to improve neurogenesis and survival of neural stem cells

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

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

Control of adult neurogenesis by programmed cell death in the mammalian brain

The presence of neural stem cells (NSCs) and the production of new neurons in the adult brain have received great attention from scientists and the public because of implications to brain plasticity and their potential use for treating currently incurable brain diseases. Adult neurogenesis is controlled at multiple levels, including proliferation, differentiation, migration, and programmed cell death (PCD). Among these, PCD is the last and most prominent process for regulating the final number of mature neurons integrated into neural circuits. PCD can be classified into apoptosis, necrosis, and autophagic cell death and emerging evidence suggests that all three may be important modes of cell death in neural stem/progenitor cells. However, the molecular mechanisms that regulate PCD and thereby impact the intricate balance between self-renewal, proliferation, and differentiation during adult neurogenesis are not well understood. In this comprehensive review, we focus on the extent, mechanism, and biological significance of PCD for the control of adult neurogenesis in the mammalian brain. The role of intrinsic and extrinsic factors in the regulation of PCD at the molecular and systems levels is also discussed. Adult neurogenesis is a dynamic process, and the signals for differentiation, proliferation, and death of neural progenitor/stem cells are closely interrelated. A better understanding of how adult neurogenesis is influenced by PCD will help lead to important insights relevant to brain health and diseases. © 2016 Ryu et al.1

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury

Differential spatial expression of peripheral olfactory neuron-derived BACE1 induces olfactory impairment by region-specific accumulation of beta-amyloid oligomer

Olfactory dysfunction is a common symptom associated with neurodegenerative diseases including Alzheimer's disease (AD). Although evidence exists to suggest that peripheral olfactory organs are involved in the olfactory dysfunction that accompanies AD pathology, the underlying mechanisms are not fully understood. As confirmed using behavioral tests, transgenic mice overexpressing a Swedish mutant form of human amyloid precursor proteins exhibited olfactory impairments prior to evidence of cognitive impairment. By measuring the expression of tyrosine hydroxylase, we observed that specific regions of the olfactory bulb (OB) in Tg2576 mice, specifically the ventral portion exhibited significant decreases in the number of dopaminergic neurons in the periglomerular regions from the early stage of AD. To confirm the direct linkage between these olfactory impairments and AD-related pathology, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1)-the initiating enzyme in A beta genesis-and beta-amyloid peptide (A beta), hallmarks of AD were analyzed. We found that an increase in BACE1 expression coincided with an elevation of amyloid-beta (A beta) oligomers in the ventral region of OB. Moreover, olfactory epithelium (OE), in particular the ectoturbinate in which axons of olfactory sensory neurons (OSNs) have direct connections with the dendrites of mitral/tufted cells in the ventral part of OB, exhibited significant decreases in both thickness and cell number even at early stages. This result suggests that A beta oligomer toxicity in the OE may have induced a decline in the number of OSNs and functional impairment of the olfactory system. We first demonstrated that disproportionate levels of regional damage in the peripheral olfactory system may be a specific symptom of AD with A beta oligomer accumulation occurring prior to damage within the CNS. This regional damage in the olfactory system early in the progression of AD may be closely related to AD-related pathological abnormality and olfactory dysfunction found in AD patients.1

A magnetically actuated microrobot for targeted neural cell delivery and selective connection of neural networks

There has been a great deal of interest in the development of technologies for actively manipulating neural networks in vitro, providing natural but simplified environments in a highly reproducible manner in which to study brain function and related diseases. Platforms for these in vitro neural networks require precise and selective neural connections at the target location, with minimal external influences, and measurement of neural activity to determine how neurons communicate. Here, we report a neuron-loaded microrobot for selective connection of neural networks via precise delivery to a gap between two neural clusters by an external magnetic field. In addition, the extracellular action potential was propagated from one cluster to the other through the neurons on the microrobot. The proposed technique shows the potential for use in experiments to understand how neurons communicate in the neural network by actively connecting neural clusters. Copyright © 2020 The Authors, some rights reserved.1