Cytoplasmic Protein Aggregates Research Articles

Protective lifelines: Tunneling nanotubes connect neurons and microglia

Tunneling nanotubes (TNTs) facilitate the exchange of intracellular cargo between cells. In this issue of Neuron, Scheiblich etal.1 reveal that TNTs selectively mediate the bidirectional transfer of cytoplasmic protein aggregates from neurons to microglia and mitochondria from microglia to neurons, thereby preserving neuronal health.

YTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner

YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N6-methyladenosine (m6A), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the m6A-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.

A safety mechanism enables tissue-specific resistance to protein aggregation during aging in C. elegans.

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

Aberrant dynein function promotes TDP-43 aggregation and upregulation of p62 in male mice harboring transgenic human TDP-43

Objective Most TDP-43 mouse models of ALS do not display cytoplasmic mislocalisation or protein aggregation of TDP-43 in spinal motor neurons in vivo. Thus, we investigated whether a combination of defective dynein with a TDP-43 mutation could trigger TDP-43 pathology. Methods Using immunohistochemical methods we examined the intracellular motor neuron pathology of the offspring of TDP-43WT and TDP-43M337V transgenic mice bred to heterozygous Loa mice, which carry an autosomal dominant mutation in dynein cytoplasmic 1 heavy chain 1 (Dync1h1). Results These mice did not exhibit TDP-43 mislocalisation in spinal motor neurons, but the expression of mutant dynein in combination with wildtype human TDP-43 resulted in p62 upregulation and TDP-43 aggregation, thus partially recapitulating the human disease. Conclusions These findings provide new insights into the possible relationship between dynein and TDP-43 and could prove useful in future studies looking to elucidate the mechanism behind the TDP-43 pathology observed in ALS.

Loss of PML nuclear bodies in familial amyotrophic lateral sclerosis-frontotemporal dementia

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders that share genetic causes and pathogenic mechanisms. The critical genetic players of ALS and FTD are the TARDBP, FUS and C9orf72 genes, whose protein products, TDP-43, FUS and the C9orf72-dipeptide repeat proteins, accumulate in form of cytoplasmic inclusions. The majority of the studies focus on the understanding of how cells control TDP-43 and FUS aggregation in the cytoplasm, overlooking how dysfunctions occurring at the nuclear level may influence the maintenance of protein solubility outside of the nucleus. However, protein quality control (PQC) systems that maintain protein homeostasis comprise a cytoplasmic and a nuclear arm that are interconnected and share key players. It is thus conceivable that impairment of the nuclear arm of the PQC may have a negative impact on the cytoplasmic arm of the PQC, contributing to the formation of the cytoplasmic pathological inclusions. Here we focused on two stress-inducible condensates that act as transient deposition sites for misfolding-prone proteins: Promyelocytic leukemia protein (PML) nuclear bodies (PML-NBs) and cytoplasmic stress granules (SGs). Upon stress, PML-NBs compartmentalize misfolded proteins, including defective ribosomal products (DRiPs), and recruit chaperones and proteasomes to promote their nuclear clearance. SGs transiently sequester aggregation-prone RNA-binding proteins linked to ALS-FTD and mRNAs to attenuate their translation. We report that PML assembly is impaired in the human brain and spinal cord of familial C9orf72 and FUS ALS-FTD cases. We also show that defective PML-NB assembly impairs the compartmentalization of DRiPs in the nucleus, leading to their accumulation inside cytoplasmic SGs, negatively influencing SG dynamics. Although it is currently unclear what causes the decrease of PML-NBs in ALS-FTD, our data highlight the existence of a cross-talk between the cytoplasmic and nuclear PQC systems, whose alteration can contribute to SG accumulation and cytoplasmic protein aggregation in ALS-FTD.

Restoring Axonal Organelle Motility and Regeneration in Cultured FUS-ALS Motoneurons through Magnetic Field Stimulation Suggests an Alternative Therapeutic Approach.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease characterized by sustained loss of neuromuscular junctions, degenerating corticospinal motoneurons and rapidly progressing muscle paralysis. Motoneurons have unique features, essentially a highly polarized, lengthy architecture of axons, posing a considerable challenge for maintaining long-range trafficking routes for organelles, cargo, mRNA and secretion with a high energy effort to serve crucial neuronal functions. Impaired intracellular pathways implicated in ALS pathology comprise RNA metabolism, cytoplasmic protein aggregation, cytoskeletal integrity for organelle trafficking and maintenance of mitochondrial morphology and function, cumulatively leading to neurodegeneration. Current drug treatments only have marginal effects on survival, thereby calling for alternative ALS therapies. Exposure to magnetic fields, e.g., transcranial magnetic stimulations (TMS) on the central nervous system (CNS), has been broadly explored over the past 20 years to investigate and improve physical and mental activities through stimulated excitability as well as neuronal plasticity. However, studies of magnetic treatments on the peripheral nervous system are still scarce. Thus, we investigated the therapeutic potential of low frequency alternating current magnetic fields on cultured spinal motoneurons derived from induced pluripotent stem cells of FUS-ALS patients and healthy persons. We report a remarkable restoration induced by magnetic stimulation on axonal trafficking of mitochondria and lysosomes and axonal regenerative sprouting after axotomy in FUS-ALS in vitro without obvious harmful effects on diseased and healthy neurons. These beneficial effects seem to derive from improved microtubule integrity. Thus, our study suggests the therapeutic potential of magnetic stimulations in ALS, which awaits further exploration and validation in future long-term in vivo studies.

Novel variants, muscle imaging, and myopathological changes in Chinese patients with VCP-related multisystem proteinopathy.

The objective of this research was to study the clinical features, genetic characteristics, muscle imaging, and muscle pathological changes of a cohort of Chinese patients with mutations in the valosin-containing protein (VCP) gene. Nine patients from seven Chinese pedigrees were recruited. Variants were detected by next-generation sequencing and confirmed by Sanger sequencing. Thigh muscle MRIs were performed in five patients. All the patients received muscle biopsies. Seven variants in VCP were identified, and two were novel. All the patients presented with adult-onset muscle weakness. The appearance of "isolated island sign" or "contra-isolated island sign" was observed in four of the five the patients on muscle MRIs. Muscle biopsies demonstrated the combination of neuropathic and myopathic changes in seven patients and muscle dystrophic changes in two patients. Notably, rimmed vacuoles and cytoplasmic VCP and p62-positive protein aggregates were observed in all the patients. Our finding of novel variants expanded the mutational spectrum of the VCP gene. This cohort of Chinese patients with VCP mutations mainly present with inclusion body myopathy with predominant limb-girdle distribution. The characteristic pattern of fatty infiltration, especially the "isolated island" and "contra-isolated island" on muscle MRI, along with rimmed vacuoles in muscle biopsy, provides valuable clues for guiding genetic diagnostic workup.

Damaged DNA Is an Early Event of Neurodegeneration in Induced Pluripotent Stem Cell-Derived Motoneurons with UBQLN2P497H Mutation.

Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein (TARDBP, also known as TDP-43) aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling.

Acid shock protein Asr induces protein aggregation to promote E. coli O157:H7 entering viable but non-culturable state under high pressure carbon dioxide stress

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr, encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state.

Autophagy in muscle regeneration: potential therapies for myopathies.

Autophagy classically functions as a physiological process to degrade cytoplasmic components, protein aggregates, and/or organelles, as a mechanism for nutrient breakdown, and as a regulator of cellular architecture. Its biological functions include metabolic stress adaptation, stem cell differentiation, immunomodulation and diseases regulation, and so on. Current researches have proved that autophagy dysfunction may contribute to the pathogenesis of some myopathies through impairment of myofibres regeneration. Studies of autophagy inhibition also indicate the importance of autophagy in muscle regeneration, while activation of autophagy can restore muscle function in some myopathies. In this review, we aim to report the mechanisms of action of autophagy on muscle regeneration to provide relevant references for the treatment of regenerating defective myopathies by regulating autophagy. Results have shown that one key mechanism of autophagy regulating the muscle regeneration is to affect the differentiation fate of muscle stem cells (MuSCs), including quiescence maintenance, activation and differentiation. The roles of autophagy (organelle/protein degradation, energy facilitation, and/or other) vary at different myogenic stages of the repair process. When the muscle is in homeostasis, basal autophagy can maintain the quiescence state and stemness of MuSCs by renewing organelle and protein. After injury, the increased autophagy flux contributes to meet biological energy demand of MuSCs during activation and proliferation. By mitochondrial remodelling, autophagy during differentiation can promote the metabolic transformation and balance mitochondrial‐mediated apoptosis signals in myoblasts. Autophagy in mature myofibres is also essential for the degradation of necrotic myofibres, and may affect the dynamics of MuSCs by affecting the secretion spectrum of myofibres or the recruitment of supporting cells. Except for myogenic cells, autophagy also plays an important role in regulating the function of non‐myogenic cells in the muscle microenvironment, which is also essential for successful muscle recovery. Autophagy can regulate the immune microenvironment during muscle regeneration through the recruitment and polarization of macrophages, while autophagy in endothelial cells can regulate muscle regeneration in an angiogenic or angiogenesis‐independent manner. Drug or nutrition targeted autophagy has been preliminarily proved to restore muscle function in myopathies by promoting muscle regeneration, and further understanding the role and mechanism of autophagy in various cell types during muscle regeneration will enable more effective combinatorial therapeutic strategies.

Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, we generated new Drosophila models and human iPS cell-derived cardiomyocytes. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

ABSTRACTIn the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

A model of the aged lung epithelium in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an age-related disorder that carries a universally poor prognosis and is thought to arise from repetitive micro injuries to the alveolar epithelium. To date, a major factor limiting our understanding of IPF is a deficiency of disease models, particularly in vitro models that can recapitulate the full complement of molecular attributes in the human condition. In this study, we aimed to develop a model that more closely resembles the aberrant IPF lung epithelium. By exposing mouse alveolar epithelial cells to repeated, low doses of bleomycin, instead of usual one-time exposures, we uncovered changes strikingly similar to those in the IPF lung epithelium. This included the acquisition of multiple phenotypic and functional characteristics of senescent cells and the adoption of previously described changes in mitochondrial homeostasis, including alterations in redox balance, energy production and activity of the mitochondrial unfolded protein response. We also uncovered dramatic changes in cellular metabolism and detected a profound loss of proteostasis, as characterized by the accumulation of cytoplasmic protein aggregates, dysregulated expression of chaperone proteins and decreased activity of the ubiquitin proteasome system. In summary, we describe an in vitro model that closely resembles the aberrant lung epithelium in IPF. We propose that this simple yet powerful tool could help uncover new biological mechanisms and assist in developing new pharmacological tools to treat the disease.

The optineurin/TIA1 pathway inhibits aberrant stress granule formation and reduces ubiquitinated TDP-43

SummaryAmyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease characterized by the formation of cytoplasmic ubiquitinated TDP-43 protein aggregates in motor neurons. Stress granules (SGs) are stress-induced cytoplasmic protein aggregates containing various neuropathogenic proteins, including TDP-43. Several studies have suggested that SGs are the initial site of the formation of pathogenic ubiquitinated TDP-43 aggregates in ALS neurons. Mutations in the optineurin (OPTN) and TIA1 genes are causative factors of familial ALS with TDP-43 aggregation pathology. We found that both OPTN depletion and ALS-associated OPTN mutations upregulated the TIA1 level in cells recovered from heat shock, and this upregulated TIA1 increased the amount of ubiquitinated TDP-43. Ubiquitinated TDP-43 induced by OPTN depletion was localized in SGs. Our study suggests that ALS-associated loss-of-function mutants of OPTN increase the amount of ubiquitinated TDP-43 in neurons by increasing the expression of TIA1, thereby promoting the aggregation of ubiquitinated TDP-43.

Molecular Mechanism of Autophagy and Its Regulation by Cannabinoids in Cancer.

Simple SummaryThis review examines the complex function of autophagy in malignancy and explores its regulation by cannabinoids in different cancers. Autophagy is an important process in the maintenance of cellular homeostasis, through the degradation and recycling of cytoplasmic constituents. The action of autophagy is highly dependent on tumour stage and type and the receptors with which ligands interact. Cannabinoids are growingly being acknowledged for their anticancer activities and are known to stimulate several mechanisms such as apoptosis and autophagy. Better understanding the mechanism of action behind autophagy and its regulation by cannabinoids will allow the development of novel cancer therapeutics.Autophagy is a “self-degradation” process whereby malfunctioned cytoplasmic constituents and protein aggregates are engulfed by a vesicle called the autophagosome, and subsequently degraded by the lysosome. Autophagy plays a crucial role in sustaining protein homeostasis and can be an alternative source of energy under detrimental circumstances. Studies have demonstrated a paradoxical function for autophagy in cancer, displaying both tumour suppressive and tumour promotive roles. In early phases of tumour development autophagy promotes cancer cell death. In later phases, autophagy enables cancer cells to survive and withstand therapy. Cannabinoids, which are derivatives of the Cannabis sativa L. plant, have shown to be associated with autophagy induction in cells. There is an emerging interest in studying the signalling pathways involved in cannabinoid-induced autophagy and their potential application in anticancer therapies. In this review, the molecular mechanisms involved in the autophagy degradation process will be discussed. This review also highlights a role for autophagy in cancer progression, with cannabinoid-induced autophagy presenting a novel strategy for anticancer therapy.

Epidermolysis Bullosa Simplex Keratinocytes Show Disturbed Mitochondrial Positioning and Activity

Keratin cytoskeletal proteins protect the epidermis against various stresses and participate in the regulation of fundamental cellular processes, such as growth, differentiation, and metabolism. The increasing complexity of keratin functions is underscored by consequences resulting from mutations in keratin 5 and keratin 14 (K14), which cause the skin-blistering disease epidermolysis bullosa simplex (EBS) by promoting the collapse of the keratin cytoskeleton into cytoplasmic protein aggregates (Kiritsi and Nyström, 2018).

SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomycescerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

Selective autophagy of cytosolic protein aggregates involves ribosome-free rough endoplasmic reticulum.

Autophagy is a degradative cellular process that can be both non-selective and selective and begins with the formation of a unique smooth double-membrane phagophore which wraps around a portion of the cytoplasm. Excess and damaged organelles and cytoplasmic protein aggregates are degraded by selective autophagy. Previously, we reported that in fed HepG2 cells, cytoplasmic aggregates of EDEM1 and surplus fibrinogen Aα-γ assembly intermediates are targets of selective autophagy receptors and become degraded by a selective autophagy called aggrephagy. Here, we show by multiple confocal immunofluorescence and colocalization panels the codistribution of cytoplasmic protein aggregates with the selective autophagy receptors p62/SQSTM1 and NBR1 and with the phagophore marker LC3, and that phagophores induced by vinblastine treatment contain complexes of protein aggregates and selective autophagy receptors. By combined serial ultrathin section analysis and immunoelectron microscopy, we found that in fed HepG2 cells, a basically ribosome-free subdomain of rough endoplasmic reticulum (RER) cisternae forms a cradle that engulfs the cytoplasmic protein aggregates. This RER subdomain appears structurally different from omegasomes formed by the RER, which were suggested to provide a membrane platform from which the phagophore is derived in starvation-induced autophagy. Taken together, our observations provide further evidence for the importance of RER subdomains as a site and membrane source for phagophore formation and show their involvement in selective autophagy.

USP10 is a critical factor for Tau-positive stress granule formation in neuronal cells

Tau aggregates in neurons of brain lesions is a hallmark pathology of tauopathies, including Alzheimer’s disease (AD). Recent studies suggest that the RNA-binding protein TIA1 initiates Tau aggregation by inducing the formation of stress granules (SGs) containing Tau. SGs are stress-inducible cytoplasmic protein aggregates containing many RNA-binding proteins that has been implicated as an initial site of multiple pathogenic protein aggregates in several neurodegenerative diseases. In this study, we found that ubiquitin-specific protease 10 (USP10) is a critical factor for the formation of Tau/TIA1/USP10-positive SGs. Proteasome inhibition or TIA1-overexpression in HT22 neuronal cells induced the formation of TIA1/Tau-positive SGs, and the formations were severely attenuated by depletion of USP10. In addition, the overexpression of USP10 without stress stimuli in HT22 cells induced TIA1/Tau/USP10-positive SGs in a deubiquitinase-independent manner. In AD brain lesions, USP10 was colocalized with Tau aggregates in the cell body of neurons. The present findings suggest that USP10 plays a key role in the initiation of pathogenic Tau aggregation in AD through SG formation.