Transformation of autophagic SQSTM1 droplets to SQSTM1-dependent P-bodies.

SQSTM1/p62 droplets play crucial roles in droplets-based macroautophagy/autophagy including selective autophagy and bulk autophagy. We observed that under several stress milieus, SQSTM1 droplets entirely colocalize with P-body markers, and these stress-induced SQSTM1 droplets contain mRNAs. We thus determined that under certain stress conditions, autophagic SQSTM1 droplets are converted to a type of enlarged P-bodies, designated SQSTM1/p62-dependent P-bodies (pd-PBs). Stress-enhanced SQSTM1 droplet formation drives the nucleation of pd-PBs through the interaction between SQSTM1 and the RNA-binding protein DDX6. Furthermore, pd-PBs sequester PYCARD, facilitating the assembly of NLRP3 inflammasomes, and in turn induce inflammation-related cytotoxicity. Our study suggests that under stress settings, autophagic SQSTM1 droplets are transformed to pd-PBs, underlining a critical role of SQSTM1 in P-body condensation.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

BACKGROUND: Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed. METHODS: We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/- mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. RESULTS IN VITRO: data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/- mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology. CONCLUSIONS: This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.

Discovery of SQSTM1/p62-dependent P-bodies that regulate the NLRP3 inflammasome.

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Blood biomarker-based classification study for neurodegenerative diseases.

As the population ages, neurodegenerative diseases are becoming more prevalent, making it crucial to comprehend the underlying disease mechanisms and identify biomarkers to allow for early diagnosis and effective screening for clinical trials. Thanks to advancements in gene expression profiling, it is now possible to search for disease biomarkers on an unprecedented scale.Here we applied a selection of five machine learning (ML) approaches to identify blood-based biomarkers for Alzheimer's (AD) and Parkinson's disease (PD) with the application of multiple feature selection methods. Based on ROC AUC performance, one optimal random forest (RF) model was discovered for AD with 159 gene markers (ROC-AUC = 0.886), while one optimal RF model was discovered for PD (ROC-AUC = 0.743). Additionally, in comparison to traditional ML approaches, deep learning approaches were applied to evaluate their potential applications in future works. We demonstrated that convolutional neural networks perform consistently well across both the Alzheimer's (ROC AUC = 0.810) and Parkinson's (ROC AUC = 0.715) datasets, suggesting its potential in gene expression biomarker detection with increased tuning of their architecture.

Gelation of cytoplasmic expanded CAG RNA repeats suppresses global protein synthesis.

RNA molecules with the expanded CAG repeat (eCAGr) may undergo sol-gel phase transitions, but the functional impact of RNA gelation is completely unknown. Here, we demonstrate that the eCAGr RNA may form cytoplasmic gel-like foci that are rapidly degraded by lysosomes. These RNA foci may significantly reduce the global protein synthesis rate, possibly by sequestering the translation elongation factor eEF2. Disrupting the eCAGr RNA gelation restored the global protein synthesis rate, whereas enhanced gelation exacerbated this phenotype. eEF2 puncta were significantly enhanced in brain slices from a knock-in mouse model and from patients with Huntington's disease, which is a CAG expansion disorder expressing eCAGr RNA. Finally, neuronal expression of the eCAGr RNA by adeno-associated virus injection caused significant behavioral deficits in mice. Our study demonstrates the existence of RNA gelation inside the cells and reveals its functional impact, providing insights into repeat expansion diseases and functional impacts of RNA phase transition.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

Dynamin-related protein 1 (Drp1) is typically known for its role in mitochondrial fission. A partial inhibition of this protein has been reported to be protective in experimental models of neurodegenerative diseases. The protective mechanism has been attributed primarily to improved mitochondrial function. Herein, we provide evidence showing that a partial Drp1-knockout improves autophagy flux independent of mitochondria. First, we characterized in cell and animal models that at low non-toxic concentrations, manganese (Mn), which causes parkinsonian-like symptoms in humans, impaired autophagy flux but not mitochondrial function and morphology. Furthermore, nigral dopaminergic neurons were more sensitive than their neighbouring GABAergic counterparts. Second, in cells with a partial Drp1-knockdown and Drp1 +/- mice, autophagy impairment induced by Mn was significantly attenuated. This study demonstrates that autophagy is a more vulnerable target than mitochondria to Mn toxicity. Furthermore, improving autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of mitochondrial fission.

A dominant-negative regulatory mechanism of SQSTM1 droplets-based autophagy.

SQSTM1/p62 is an autophagy receptor, forming droplets to sequester intracellular polyubiquitinated cargo and mediate its delivery for autophagic clearance. SQSTM1 droplets can function as platforms to allow the formation of autophagosomes at their surfaces. It would be interesting to understand how SQSTM1-droplet formation is regulated. We have shown that inflammatory toxicity induces SQSTM1 cleavage by CASP6 at a novel cleavage site, D256. The C-terminal cleavage product is unlikely to be functional, because it is hardly detectable, possibly due to its rapid turnover. The SQSTM1 N-terminal cleavage product (SQSTM1-N) exerts a dominant-negative effect on SQSTM1-droplet production, in turn attenuating SQSTM1 droplets-based autophagosome formation. Our study suggests that the CASP6-SQSTM1 axis negatively regulates SQSTM1 droplets-based autophagy under certain stress conditions.

The caspase-6-p62 axis modulates p62 droplets based autophagy in a dominant-negative manner.

SQSTM1/p62, as a major autophagy receptor, forms droplets that are critical for cargo recognition, nucleation, and clearance. p62 droplets also function as liquid assembly platforms to allow the formation of autophagosomes at their surfaces. It is unknown how p62-droplet formation is regulated under physiological or pathological conditions. Here, we report that p62-droplet formation is selectively blocked by inflammatory toxicity, which induces cleavage of p62 by caspase-6 at a novel cleavage site D256, a conserved site across human, mouse, rat, and zebrafish. The N-terminal cleavage product is relatively stable, whereas the C-terminal product appears undetectable. Using a variety of cellular models, we show that the p62 N-terminal caspase-6 cleavage product (p62-N) plays a dominant-negative role to block p62-droplet formation. In vitro p62 phase separation assays confirm this observation. Dominant-negative regulation of p62-droplet formation by caspase-6 cleavage attenuates p62 droplets dependent autophagosome formation. Our study suggests a novel pathway to modulate autophagy through the caspase-6-p62 axis under certain stress stimuli.

Degradation of lipid droplets by chimeric autophagy-tethering compounds.

Degrading pathogenic proteins by degrader technologies such as PROTACs (proteolysis-targeting chimeras) provides promising therapeutic strategies, but selective degradation of non-protein pathogenic biomolecules has been challenging. Here, we demonstrate a novel strategy to degrade non-protein biomolecules by autophagy-tethering compounds (ATTECs), using lipid droplets (LDs) as an exemplar target. LDs are ubiquitous cellular structures storing lipids and could be degraded by autophagy. We hypothesized that compounds interacting with both the LDs and the key autophagosome protein LC3 may enhance autophagic degradation of LDs. We designed and synthesized such compounds by connecting LC3-binding molecules to LD-binding probes via a linker. These compounds were capable of clearing LDs almost completely and rescued LD-related phenotypes in cells and in two independent mouse models with hepatic lipidosis. We further confirmed that the mechanism of action of these compounds was mediated through LC3 and autophagic degradation. Our proof-of-concept study demonstrates the capability of degrading LDs by ATTECs. Conceptually, this strategy could be applied to other protein and non-protein targets.

Enhanced lysosomal function is critical for paclitaxel resistance in cancer cells: reversed by artesunate.

The mechanism underlying the resistance of cancer cells to chemotherapeutic drug varies with different cancer cells. Recent evidence shows that lysosomal function is associated with drug resistance of cancer cells. Artesunate, a derivative of artemisinin, displays broad antitumor activity and direct cytotoxicity on various tumor cells. Our previous study shows that artesunate increases autophagosome accumulation, while significantly decreases autolysosome number in cancer cells, suggesting that artesunate might impair the lysosomal function. In this study, we investigated the effects of artesunate on lysosomal function and its relationship with chemotherapeutic drug resistance in cancer cells. We found that the lysosomal function was significantly enhanced in two drug-resistant (A549/TAX and A549/DDP) cells. Furthermore, we showed that the enhanced lysosomal function by overexpression of transcription factor EB (TFEB) significantly increased MCF-7 cells resistance to doxorubicin (DOX), whereas the decreased lysosomal function by TFEB-knockdown or lysosome inhibitor chloroquine increased MCF-7 cells sensitivity to DOX. Treatment of A549/TAX cells with artesunate (2.5-50 µM) dose-dependently inhibited lysosomal function and the clearance of dysfunctional mitochondria, and induced cell apoptosis. Moreover, we demonstrated that artesunate exerted more potent inhibition on the resistant (A549/TAX and MCF-7/ADR) cells with higher activity of lysosomal function. Our results suggest that artesunate or other inhibitors of lysosomal function would be potential in the treatment of cancer cells with drug resistance caused by the enhanced lysosomal function.

Genetic networks in Parkinson's and Alzheimer's disease.

Parkinson's disease (PD) and Alzheimer's disease (AD) are the most common neurodegenerative diseases and there is increasing evidence that they share common physiological and pathological links. Here we have conducted the largest network analysis of PD and AD based on their gene expressions in blood to date. We identified modules that were not preserved between disease and healthy control (HC) networks, and important hub genes and transcription factors (TFs) in these modules. We highlighted that the PD module not preserved in HCs was associated with insulin resistance, and HDAC6 was identified as a hub gene in this module which may have the role of influencing tau phosphorylation and autophagic flux in neurodegenerative disease. The AD module associated with regulation of lipolysis in adipocytes and neuroactive ligand-receptor interaction was not preserved in healthy and mild cognitive impairment networks and the key hubs TRPC5 and BRAP identified as potential targets for therapeutic treatments of AD. Our study demonstrated that PD and AD share common disrupted genetics and identified novel pathways, hub genes and TFs that may be new areas for mechanistic study and important targets in both diseases.

Histone H3F3/H3.3 chaperone DAXX converts to modulate SQSTM1 phase condensation for NFE2L2 activation.

Macroautophagy/autophagy cargo receptor SQSTM1/p62 puncta or clustering formation is critical for its function in cargo recognition and LC3 interaction. Evidence suggests that SQSTM1 puncta formation is a process of liquid-liquid phase separation. It is poorly understood how SQSTM1 liquid-liquid phase separation is regulated. We found that cytoplasmic DAXX enhances SQSTM1 puncta formation, and further demonstrated that DAXX drives SQSTM1 liquid phase condensation through increasing SQSTM1 oligomerization. DAXX promotes SQSTM1 recruitment of KEAP1, subsequently activating an NFE2L2/NRF2-mediated stress response. This study suggests a new mechanism of SQSTM1 phase condensation by a protein-protein interaction, and indicates that cytoplasmic DAXX can play a role to regulate redox homeostasis.

Bim contributes to the progression of Huntington's disease-associated phenotypes.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin (HTT) protein. Mutant HTT (mHTT) toxicity is caused by its aggregation/oligomerization. The striatum is the most vulnerable region, although all brain regions undergo neuronal degeneration in the disease. Here we show that the levels of Bim, a BH3-only protein, are significantly increased in HD human post-mortem and HD mouse striata, correlating with neuronal death. Bim reduction ameliorates mHTT neurotoxicity in HD cells. In the HD mouse model, heterozygous Bim knockout significantly mitigates mHTT accumulation and neuronal death, ameliorating disease-associated phenotypes and lifespan. Therefore, Bim could contribute to the progression of HD.

Lowering Mutant Huntingtin Levels and Toxicity: Autophagy-Endolysosome Pathways in Huntington's Disease.

Huntington's disease (HD) is a monogenetic neurodegenerative disease, which serves as a model of neurodegeneration with protein aggregation. Autophagy has been suggested to possess a great value to tackle protein aggregation toxicity and neurodegenerative diseases. Current studies suggest that autophagy-endolysosomal pathways are critical for HD pathology. Here we review recent advancement in the studies of autophagy and selective autophagy relating HD. Restoration of autophagy flux and enhancement of selective removal of mutant huntingtin/disease-causing protein would be effective approaches towards tackling HD as well as other similar neurodegenerative disorders.

Exosome release and neuropathology induced by α-synuclein: new insights into protective mechanisms of Drp1 inhibition.

Targeting alpha-synuclein (α-syn) as a therapeutic strategy for Parkinson's disease (PD) has been intensively pursued largely due to its well-recognized pathogenic role. Since its discovery as the first familial link to PD over two decades ago, this protein has been associated with multiple neurotoxic mechanisms, such as mitochondrial dysfunction and impaired autophagic flux. We report here that blocking dynamin-related protein 1 (Drp1) improved both mitochondrial function and autophagic flux in experimental models of α-syn. Using rat dopaminergic neuronal cells with inducible wild-type human α-syn, we observed excessive mitochondrial fragmentation and increased Drp1 levels 48 h after gene induction. Functionally, these cells exhibited lower mitochondrial membrane potential, reduced ATP production rate and mitochondrial spare respiratory capacity, as well as increased levels of mitochondrial reactive oxygen species. To evaluate the protective role of Drp1 inhibition, we used three complementary approaches: gene silencing mediated by siRNA, overexpression of Drp1-dominant negative and the small molecule mitochondrial division inhibitor-1 (mdivi-1). Both morphological and functional defects induced by α-syn were attenuated by these strategies. Importantly, Drp1 inhibition reduced proteinase K-resistant α-syn aggregates. Based on that observation, we investigated the involvement of autophagy. Through a combination of stable autophagy reporter cells and immunoreactivity for LC3 and p62 in neuronal cells with either α-syn overexpression or treatment of human α-syn preformed fibrils (PFF), we observed that Drp1 inhibition abolished autophagic impairment induced by α-syn. Consistent with its role in improving autophagy function, Drp1 inhibition reduced exosome release and spread of α-syn pathology from neurons to neurons and from microglia to neurons. In summary, this study highlights new insights that Drp1 inhibition confers neuroprotection through both mitochondrial and autophagy-lysosomal pathways, further strengthening the therapeutic potential of targeting Drp1.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response.

Autophagy cargo recognition and clearance are essential for intracellular protein quality control. SQSTM1/p62 sequesters intracellular aberrant proteins and mediates cargo delivery for their selective autophagic degradation. The formation of p62 non-membrane-bound liquid compartments is critical for its function as a cargo receptor. The regulation of p62 phase separation/condensation has yet been poorly characterised. Using an unbiased yeast two-hybrid screening and complementary approaches, we found that DAXX physically interacts with p62. Cytoplasmic DAXX promotes p62 puncta formation. We further elucidate that DAXX drives p62 liquid phase condensation by inducing p62 oligomerisation. This effect promotes p62 recruitment of Keap1 and subsequent Nrf2-mediated stress response. The present study suggests a mechanism of p62 phase condensation by a protein interaction, and indicates that DAXX regulates redox homoeostasis, providing a mechanistic insight into the prosurvival function of DAXX.

Mitochondrial integrity in neurodegeneration.

The mitochondrion is a unique organelle with a diverse range of functions. Mitochondrial dysfunction is a key pathological process in several neurodegenerative diseases. Mitochondria are mostly important for energy production; however, they also have roles in Ca2+ homeostasis, ROS production, and apoptosis. There are two major systems in place, which regulate mitochondrial integrity, mitochondrial dynamics, and mitophagy. These two processes remove damaged mitochondria from cells and protect the functional mitochondrial population. These quality control systems often become dysfunctional during neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, causing mitochondrial dysfunction and severe neurological symptoms.