Autophagy Deficits Research Articles

SUMOylation modulates mitochondrial dynamics in an in vitro rotenone model of Parkinson's disease

SUMOylation is a post-translational modification essential for various biological processes. SUMO proteins bind to target substrates in a three-step enzymatic pathway, which is rapidly reversible by the action of specific proteases, known as SENPs. Studies have shown that SUMOylation is dysregulated in several human disorders, including neurodegenerative diseases that are characterized by the progressive loss of neurons, mitochondrial dysfunction, deficits in autophagy, and oxidative stress. Considering the potential neuroprotective roles of SUMOylation, the aim of this study was to investigate the effects of SENP3 knockdown in H4 neuroglioma cells exposed to rotenone, an in vitro model of cytotoxicity that mimics dopaminergic loss in Parkinson's disease (PD). The current data show that SENP3 knockdown increases SUMO-2/3 conjugates, which is accompanied by reduced levels of the mitochondrial fission protein Drp1 and increased levels of the mitochondrial fusion protein OPA1. Of high interest, SENP3 knockdown prevented rotenone-induced superoxide production and cellular death. Taken together, these findings highlight the importance of SUMOylation in maintaining mitochondrial homeostasis and the neuroprotective potential of this modification in PD.

Oleanonic acid ameliorates mutant Aβ precursor protein-induced oxidative stress, autophagy deficits, ferroptosis, mitochondrial damage, and ER stress in vitro

Accumulation in the brain of amyloid-β (Aβ), derived from cleavage of Aβ precursor protein (APP), is a hallmark of Alzheimer's disease (AD). Oleanonic acid (OA), a phytochemical from several plants, has proven anti-inflammatory effects, but its role in AD remains unknown. Here we found that OA reduced APP expression and inhibited oxidative stress via Nrf2/HO-1 signaling in SH-SY5Y neuroblastoma cells stably overexpressing APP. OA suppressed phosphorylated mTOR but increased autophagy markers ATG5 and LC3-II. Moreover, OA rescued ferroptosis-related factors GPX4, NCOA, and COX2 and ER stress markers GRP78, CHOP, and three main induction pathways of ER stress including IRE1/XBP1s, PERK/EIF2α, and ATF6. OA alleviated mitochondrial damage through MFN1, MFN2, OPA1, FIS1, and DRP1. Furthermore, OA upregulated GDF11 expression and downregulated phosphorylation of ErbB4 and TrkB without affecting BDNF levels. Thus, OA might protect neurons from APP-induced neurotoxicity by inhibiting oxidative stress, autophagy deficits, ferroptosis, mitochondrial damage, and ER stress in AD, providing a new promising therapeutic strategy in patients with AD.

The Drosophila Nesprin-1 homolog MSP300 is required for muscle autophagy and proteostasis.

Nesprin proteins, which are components of the linker of nucleoskeleton and cytoskeleton (LINC) complex, are located within the nuclear envelope and play prominent roles in nuclear architecture. For example, LINC complex proteins interact with both chromatin and the cytoskeleton. Here, we report that the Drosophila Nesprin MSP300 has an additional function in autophagy within larval body wall muscles. RNAi-mediated MSP300 knockdown in larval body wall muscles resulted in defects in the contractile apparatus, muscle degeneration and defective autophagy. In particular, MSP300 knockdown caused accumulation of cytoplasmic aggregates that contained poly-ubiquitylated cargo, as well as the autophagy receptor ref(2)P (the fly homolog of p62 or SQSTM) and Atg8a. Furthermore, MSP300 knockdown larvae expressing an mCherry-GFP-tagged Atg8a transgene exhibited aberrant persistence of the GFP signal within these aggregates, indicating failure of autophagosome maturation. These autophagy deficits were similar to those exhibited by loss of the endoplasmic reticulum (ER) fusion protein Atlastin (Atl), raising the possibility that Atl and MSP300 might function in the same pathway. In support of this possibility, we found that a GFP-tagged MSP300 protein trap exhibited extensive localization to the ER. Alteration of ER-directed MSP300 might abrogate important cytoskeletal contacts necessary for autophagosome completion.

NLRP3-mediated autophagy dysfunction links gut microbiota dysbiosis to tau pathology in chronic sleep deprivation.

Emerging evidence indicates that sleep deprivation (SD) can lead to Alzheimer's disease (AD)-related pathological changes and cognitive decline. However, the underlying mechanisms remain obscure. In the present study, we identified the existence of a microbiota-gut-brain axis in cognitive deficits resulting from chronic SD and revealed a potential pathway by which gut microbiota affects cognitive functioning in chronic SD. Our findings demonstrated that chronic SD in mice not only led to cognitive decline but also induced gut microbiota dysbiosis, elevated NLRP3 inflammasome expression, GSK-3β activation, autophagy dysfunction, and tau hyperphosphorylation in the hippocampus. Colonization with the "SD microbiota" replicated the pathological and behavioral abnormalities observed in chronic sleep-deprived mice. Remarkably, both the deletion of NLRP3 in NLRP3 -/- mice and specific knockdown of NLRP3 in the hippocampus restored autophagic flux, suppressed tau hyperphosphorylation, and ameliorated cognitive deficits induced by chronic SD, while GSK-3β activity was not regulated by the NLRP3 inflammasome in chronic SD. Notably, deletion of NLRP3 reversed NLRP3 inflammasome activation, autophagy deficits, and tau hyperphosphorylation induced by GSK-3β activation in primary hippocampal neurons, suggesting that GSK-3β, as a regulator of NLRP3-mediated autophagy dysfunction, plays a significant role in promoting tau hyperphosphorylation. Thus, gut microbiota dysbiosis was identified as a contributor to chronic SD-induced tau pathology via NLRP3-mediated autophagy dysfunction, ultimately leading to cognitive deficits. Overall, these findings highlight GSK-3β as a regulator of NLRP3-mediated autophagy dysfunction, playing a critical role in promoting tau hyperphosphorylation.

Aerobic exercise attenuates autophagy-lysosomal flux deficits by ADRB2/β2-adrenergic receptor-mediated V-ATPase assembly factor VMA21 signaling in APP-PSEN1/PS1 mice

ABSTRACT Growing evidence suggests that macroautophagy/autophagy-lysosomal pathway deficits contribute to the accumulation of amyloid-β (Aβ) in Alzheimer disease (AD). Aerobic exercise (AE) has long been investigated as an approach to delay and treat AD, although the exact role and mechanism are not well known. Here, we revealed that AE could reverse autophagy-lysosomal deficits via activation of ADRB2/β2-adrenergic receptor, leading to significant attenuation of amyloid-β pathology in APP-PSEN1/PS1 mice. Molecular mechanism research found that AE could reverse autophagy deficits by upregulating the AMP-activated protein kinase (AMPK)-MTOR (mechanistic target of rapamycin kinase) signaling pathway. Moreover, AE could reverse V-ATPase function by upregulating VMA21 levels. Inhibition of ADRB2 by propranolol (antagonist, 30 μM) blocked AE-attenuated Aβ pathology and cognitive deficits by inhibiting autophagy-lysosomal flux. AE may mitigate AD via many pathways, while ADRB2-VMA21-V-ATPase could improve cognition by enhancing the clearance of Aβ through the autophagy-lysosomal pathway, which also revealed a novel theoretical basis for AE attenuating pathological progression and cognitive deficits in AD.

Metformin rescues migratory deficits of cells derived from patients with periventricular heterotopia.

Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells (hNPCs) derived from PH patients with a DCHS1 or FAT4 mutation as well as isogenic lines had altered migratory dynamics when grafted in the mouse brain. The affected migration was linked to altered autophagy as observed in vivo with an electron microscopic analysis of grafted hNPCs, a Western blot analysis of cortical organoids, and time-lapse imaging of hNPCs in the presence of bafilomycin A1. We further show that deficits in autophagy resulted in the accumulation of paxillin, a focal adhesion protein involved in cell migration. Strikingly, a single-cell RNA-seq analysis of hNPCs revealed similar expression levels of autophagy-related genes. Bolstering AMPK-dependent autophagy by metformin, an FDA-approved drug, promoted migration of PH patients-derived hNPCs. Our data indicate that transcription-independent homeostatic modifications in autophagy contributed to the defective migratory behavior of hNPCs in vivo and suggest that modulating autophagy in hNPCs might rescue neuronal migration deficits in some forms of PH.

Age-related deficits in retinal autophagy following intraocular pressure elevation in autophagy reporter mouse model

This study quantified age-related changes to retinal autophagy using the CAG-RFP-EGFP-LC3 autophagy reporter mice and considered how aging impacts autophagic responses to acute intraocular pressure (IOP) stress. IOP was elevated to 50 mm Hg for 30 minutes in 3-month-old and 12-month-old CAG-RFP-EGFP-LC3 (n = 7 per age group) and Thy1-YFPh transgenic mice (n = 3 per age group). Compared with younger eyes, older eyes showed diminished basal autophagy in the outer retina, while the inner retina was unaffected. Autophagic flux (red:yellow puncta ratio) was elevated in the inner plexiform layer. Three days following IOP elevation, older eyes showed poorer functional recovery, most notably in ganglion cell responses compared to younger eyes (12 months old: −33.4 ± 5.3% vs. 3 months mice: −13.4 ± 4.5%). This paralleled a reduced capacity to upregulate autophagic puncta volume in the inner retina in older eyes, a response that was seen in younger eyes. Age-related decline in basal and stress-induced autophagy in the retina is associated with greater retinal ganglion cells' susceptibility to IOP elevation.

Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration.

Although microglial activation is widely found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the underlying mechanism(s) are poorly understood. Here, using human-induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harboring the most common ALS/FTD mutation (C9orf72, mC9-MG), gene-corrected isogenic controls (isoC9-MG), and C9orf72 knockout hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated immune response upon stimulation with lipopolysaccharide. Analysis of the C9ORF72 interactome revealed that C9ORF72 interacts with regulators of autophagy and functional studies showed impaired initiation of autophagy in mC9-MG and C9KO-MG. Coculture studies with motor neurons (MNs) demonstrated that the autophagy deficit in mC9-MG drives increased vulnerability of mC9-MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated both cell-autonomous functional deficits in hiPSC-MG and MN death in MG-MN coculture. Together, these findings reveal an important role for C9ORF72 in regulating immune homeostasis and identify dysregulation in myeloid cells as a contributor to neurodegeneration in ALS/FTD.

Lactobacillus plantarum postbiotics trigger AMPK-dependent autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome activation.

We previously found that Lactobacillus plantarum (LP)-derived postbiotics protected animals against Salmonella infection, but the molecular mechanism remains obscure. This study clarified the mechanisms from the perspective of autophagy. Intestinal porcine epithelial cells (IPEC-J2) were pretreated with LP-derived postbiotics (the culture supernatant, LPC; or heat-killed bacteria, LPB), and then challenged with Salmonella enterica Typhimurium (ST). Results showed that LP postbiotics markedly triggered autophagy under ST infection, as indicated by the increased LC3 and Beclin1 and the decreased p62 levels. Meanwhile, LP postbiotics (particularly LPC) exhibited a strong capacity of inhibiting ST adhesion, invasion and replication. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) led to a significant decrease of autophagy and the aggravated infection, indicating the importance of autophagy in LP postbiotics-mediated Salmonella elimination. LP postbiotics (especially LPB) significantly suppressed ST-induced inflammation by modulating inflammatory cytokines (the increased interleukin (IL)-4 and IL-10, and decreased tumor necrosis factor-α (TNF), IL-1β, IL-6 and IL-18). Furthermore, LP postbiotics inhibited NOD-like receptor protein 3 (NLRP3)inflammasome activation, as evidenced by the decreased levels of NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Deficits in autophagy resulted in an increase of inflammatory response and inflammasome activation. Finally, we found that both LPC and LPB triggered AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy, and this was further confirmed by AMPK RNA interference. The intracellular infection and NLRP3 inflammasome were aggravated after AMPK knockdown. In summary, LP postbiotics trigger AMPK-mediated autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome in IPEC-J2 cells. Our findings highlight the effectiveness of postbiotics, and provide a new strategy for preventing Salmonella infection.

The role of PKC/PKR in aging, Alzheimer's disease, and perioperative neurocognitive disorders.

BackgroundThe incidence of perioperative neurocognitive disorders (PNDs) is reportedly higher in older patients. Mitochondrial and synaptic dysfunctions have consistently been demonstrated in models of aging and neurodegenerative diseases; nonetheless, their role in PND is not well understood.MethodsThe Morris water maze and elevated plus maze tests were used to assess the learning and memory abilities of both C57BL/6 and 3×Tg-AD mice of different ages (8 and 18 months). PND was induced by laparotomy in C57BL/6 mice and 3×Tg-AD mice (8 months old). Markers associated with neuroinflammation, mitochondrial function, synaptic function, and autophagy were assessed postoperatively. The roles of protein kinase C (PKC) and double-stranded RNA-dependent protein kinase (PKR) were further demonstrated by using PKC-sensitive inhibitor bisindolylmaleimide X (BIMX) or PKR−/− mice.ResultsSignificant cognitive impairment was accompanied by mitochondrial dysfunction and autophagy inactivation in both aged C57BL/6 and 3×Tg-AD mice. Laparotomy induced a significant neuroinflammatory response and synaptic protein loss in the hippocampus. Cognitive and neuropathological changes induced by aging or laparotomy were further exacerbated in 3×Tg-AD mice. Deficits in postoperative cognition, hippocampal mitochondria, autophagy, and synapse were significantly attenuated after pharmacological inhibition of PKC or genetic deletion of PKR.ConclusionsOur findings suggest similar pathogenic features in aging, Alzheimer's disease, and PND, including altered mitochondrial homeostasis and autophagy dysregulation. In addition, laparotomy may exacerbate cognitive deficits associated with distinct neuronal inflammation, mitochondrial dysfunction, and neuronal loss independent of genetic background. The dysregulation of PKC/PKR activity may participate in the pathogenesis of these neurodegenerative diseases.

Update on Melasma-Part I: Pathogenesis.

Melasma is a multifactorial dyschromia that results from exposure to external factors (such as solar radiation) and hormonal factors (such as sex hormones and pregnancy), as well as skin inflammation (such as contact dermatitis and esthetic procedures), in genetically predisposed individuals. Beyond hyperfunctional melanocytes, skin with melasma exhibits a series of structural and functional alterations in the epidermis, basement membrane, and upper dermis that interact to elicit and sustain a focal hypermelanogenic phenotype. Evolution in the knowledge of the genetic basis of melasma and the cutaneous response to solar radiation, as well as the roles of endocrine factors, antioxidant system, endothelium proliferation, fibroblast senescence, mast cell degranulation, autophagy deficits of the melanocyte, and the paracrine regulation of melanogenesis, will lead to the development of new treatments and preventive strategies. This review presents current knowledge on these aspects of the pathogenesis of melasma and discusses the effects of specific treatments and future research on these issues.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling

BackgroundAutophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease (AD). This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.MethodsThe primary hippocampal neurons, N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy, which was analysed by Student's two-tailed t-test. The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1 (mTORC1) activity and the vacuolar H+-ATPase (v-ATPase) activity, respectively, which were analysed by One‐way ANOVA with post hoc tests. The Western blotting, co-immunoprecipitation and immunofluorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations, as analysed by Student's two-tailed t-test or One‐way ANOVA with post hoc tests. The autophagosome formation was detected by immunofluorescence staining and transmission electron microscopy. The amino acids (AA) levels were detected by high performance liquid chromatography (HPLC).ResultsWe observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits. Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1 (PRD-TIA1) and this association significantly increased the intercellular level of amino acids (Leucine, P = 0.0038; Glutamic acid, P = 0.0348; Alanine, P = 0.0037; Glycine, P = 0.0104), with concordant upregulation of mTORC1 activity [phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), P < 0.0001; phosphorylated 70 kDa ribosomal protein S6 kinase 1 (p-p70S6K1), P = 0.0001, phosphorylated unc-51-like autophagy-activating kinase 1 (p-ULK1), P = 0.0015] and inhibition of autophagosome formation [microtubule-associated protein light chain 3 II (LC3 II), P = 0.0073; LC3 puncta, P < 0.0001]. As expected, this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation. Importantly, we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1, downregulating the endogenous TIA1 expression by shRNA, or downregulating tau protein level by a small proteolysis targeting chimera (PROTAC) could remarkably attenuate tau-induced autophagy impairment.ConclusionsOur findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway, and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treatment and that of related tauopathies.

Selective autophagy plays a protective role against acute and age- related retinal degeneration

Objective. Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status, this context requires a tightly-regulated housekeeping system that includes selective mitochondrial autophagy. We want to assess the effects of selective autophagy deficit or induction in the retina given an AMD-like paradigm. Materials and methods. Eyes from Ambra1+/+, Ambra1+/gt and mito-QC mice were analysed using flatmount or cryosection immunostaining. Sodium iodate (SI) was used as a model of AMD-like damage and Urolithin A (UA) as a mitophagy inducer. ARPE-19 human cells were used as an in vitro model and analysed by immunostaining, flow cytometry and RT-qPCR. Bioinformatic analysis of public human datasets was also performed. Results. Ambra1+/gt autophagy-deficient mice present alterations in the RPE, similar to those observed in human AMD patients, such as abnormal morphology or lipofuscin accumulation, which appear in an age-dependent manner. Furthermore, Ambra1+/gt mice are more sensitive to acute SI-induced retinal degeneration than their Ambra+/+ littermates. UA induced mitophagy in vivo and prevented degeneration both in the neuroretina and RPE. This amelioration was also associated with decreased lipid peroxidation, gliosis and increased photoreceptor survival. In vitro, inhibition of mitophagy, or general macroautophagy, abolished this rescue. Conclusions. Selective autophagy plays a protective role in the retina and can be exploited to preserve vision in physiological or pathological conditions.

Autophagy Regulation Influences β-Amyloid Toxicity in Transgenic Caenorhabditis elegans.

Alzheimer’s disease (AD) is a progressive, neurodegenerative disease characterized by the accumulation of amyloid-beta (Aβ) proteins in the form of plaques that cause a proteostasis imbalance in the brain. Several studies have identified autophagy deficits in both AD patients and AD animal models. Here, we used transgenic Caenorhabditis elegans to study the relationship between autophagy flux and Aβ. We labeled autophagosomes with an advanced fluorescence reporter system, and used this to observe that human Aβ expression caused autophagosome accumulation in C. elegans muscle. The autophagy-related drugs chloroquine and 3-MA were employed to investigate the relationship between changes in autophagic flux and the toxicity of Aβ expression. We found that reducing autophagosome accumulation delayed Aβ-induced paralysis in the CL4176 strain of C. elegans, and alleviated Aβ-induced toxicity, thus having a neuroprotective effect. Finally, we used RNA-sequencing and proteomics to identify genes whose expression was affected by Aβ aggregation in C. elegans. We identified a series of enriched autophagy-related signal pathways, suggesting that autophagosome accumulation impairs Aβ protein homeostasis in nematodes. Thus, maintaining normal autophagy levels appears to be important in repairing the protein homeostasis imbalance caused by Aβ expression.

Microglial NF-\u03baB drives tau spreading and toxicity in a mouse model of tauopathy

Activation of microglia is a prominent pathological feature in tauopathies, including Alzheimer’s disease. How microglia activation contributes to tau toxicity remains largely unknown. Here we show that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, activated by tau, drives microglial-mediated tau propagation and toxicity. Constitutive activation of microglial NF-κB exacerbated, while inactivation diminished, tau seeding and spreading in young PS19 mice. Inhibition of NF-κB activation enhanced the retention while reduced the release of internalized pathogenic tau fibrils from primary microglia and rescued microglial autophagy deficits. Inhibition of microglial NF-κB in aged PS19 mice rescued tau-mediated learning and memory deficits, restored overall transcriptomic changes while increasing neuronal tau inclusions. Single cell RNA-seq revealed that tau-associated disease states in microglia were diminished by NF-κB inactivation and further transformed by constitutive NF-κB activation. Our study establishes a role for microglial NF-κB signaling in mediating tau spreading and toxicity in tauopathy.

Long-Term Social Isolation-Induced Autophagy Inhibition and Cell Senescence Aggravate Cognitive Impairment in D(+)Galactose-Treated Male Mice.

Aging is associated with physiological and pathological changes and presents health complications, such as dementia. Isolation has also been associated with the experience of growing old. Both have been linked individually to the incidence of cognitive decline. In this present study, the effects of these two phenomena have been looked at in animal models where aging was induced with D(+)Galactose in mice who underwent long-term post-weaned social isolation (L-PWSI). Assessing cognitive function using Y-maze, Morris water maze (MWM), and passive avoidance tests (PATs) confirmed that cognition is impaired in either of the treatments but worsened when the D(+)Galactose mice were subjected to L-PWSI. Moreover, a synaptic protein, PSD95, and dendritic spines density were significantly reduced in the L-PWSI and D(+)Galactose-treated mice. Our previous study revealed that autophagy deficit is involved in cognitive impairment in the L-PWSI model. Here, we first report the inhibited cell cycle in L-PWSI, combined with the decreased autophagy, aggravates cognitive impairment in D(+)Galactose-treated mice. Beyond these, the autophagy and cell cycle mechanisms that link isolation and aging have been explored. The close association between isolation and aging in humans is very real and needs much research attention going forward for possible therapeutic interventions.

Cornel Iridoid Glycoside and Its Effective Component Regulate ATPase Vps4A/JNK to Alleviate Autophagy Deficit with Autophagosome Accumulation.

Improving autophagy-lysosome fusion has been considered a key method in the treatment of Alzheimer's disease (AD). Cornel iridoid glycoside (CIG) is extracted from Cornus officinalis and has been shown to promote the clearance of tau oligomers via the autophagy pathway. However, the mechanisms of CIG on autophagy deficits are not understood. Here, we found autophagy deficit and tau aggregation in the brains of P301S tau transgenic mice and MAPT cells edited using CRISPR-Cas9 technology. CIG decreased tau aggregation and alleviated autophagic markers involving the JNK/Beclin-1 signaling pathway which demonstrated CIG that might enhance lysosome formation by upregulating ATPase Vps4A expression. Knocking down VPS4A increased autophagosome accumulation and attenuated the effect of CIG on p62. In addition, CIG had no effect on tau oligomers but still inhibited the level of tau monomer in VPS4A knockout cells. The effective component (Sweroside, SWE) of CIG attenuated tau oligomers accumulation and increased Vps4A level but not CHMP2B. SWE could not change the level of tau oligomers in VPS4A knockout cells. In conclusion, CIG suppressed autophagosome accumulation by regulating the ATPase Vps4A/JNK. SWE is a core of active factors of CIG in Vps4A regulation. These findings suggest CIG may be a potential drug in AD treatment.

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice.Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI.Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage.Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.

Distinct subcellular autophagy impairments in induced neurons from patients with Huntington's disease

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics.Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy.In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

Fibroblast Growth Factor 21 and Autophagy Modulation Ameliorates Amyloid β‐Induced Alzheimer Disease Pathology in Rats

AbstractBackgroundAlzheimer's disease (AD) is a multifactorial neurodegenerative condition and the most common cause of its initiation is accumulation of oligomeric amyloid beta1‐42 (Aβ1‐42). In recent past, several studies have shown autophagy deficits in AD may resulted accumulation of misfolded protein, Aβ1‐42 and phosphorylated tau (ptau). Fibroblast growth factor 21 (FGF21), a metabolic hormone, has shown strong neuroprotective efficacy via increasing autophagic flux in AD. Therefore, this study was designed to investigate the synergistic neuroprotective efficacy of lentiviral FGF21 gene (LV‐FGF21) delivery and rapamycin‐autophagy modulator in Aβ1‐42 induced AD in rats.MethodLV‐FGF21 was administered 4weeks before inducing AD by oligomeric Aβ1‐42 into the lateral cerebral ventricles. Rats were divided into control, sham group administered with PBS intracerebroventricularly (ICV), ICV‐Aβ1‐42 group, ICV‐Aβ1‐42 group pre‐treated with LV‐FGF21, and pre‐treated LV‐FGF21 group combined with rapamycin. Morris water maze (MWM), ELISA assay for measuring protein levels of Aβ1‐42, ptau, soluble amyloid precursor protein (sAPP) and beta‐site APP cleaving enzyme 1 (BACE1), oxidative stress measurement, mRNA expression and immunofluorescence analysis were performed after treatment completion.ResultAβ1‐42 oligomer formation was confirmed by FESEM analysis. After 14 days, MWM analysis showed that the combination group considerably restored the cognitive ability while attenuated the level of Aβ1‐42, ptau, sAPP, and BACE1. Moreover, oxidative stress parameters considerably improved for the combined treated group as compared with ICV‐Aβ1‐42 group. Also, mRNA expression of main autophagy mediators (beclin‐1, LAMP‐2, LC3, ATF4, p62/SQSTM1) reflected positive outcome for the combination treatment group.ConclusionThe findings of the present study confirmed the neuroprotective potential of FGF21 and rapamycin in debilitating AD pathology in rats.