Degradation Of Protein Aggregates Research Articles

Upregulated pexophagy limits the capacity of selective autophagy

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson’s Disease and Huntington’s Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

Autophagy Receptor-Inspired Antibody-Fusion Proteins for Targeted Intracellular Degradation.

Autophagy is responsible for the degradation of large intracellular contents, such as unwanted protein aggregates and organelles. Impaired autophagy can therefore lead to the accumulation of pathological aggregates, correlating with aging and neurodegenerative diseases. However, a broadly applicable methodology is not available for the targeted degradation of protein aggregates or organelles in mammalian cells. Herein, we developed a series of autophagy receptor-inspired targeting chimeras (AceTACs) that can induce the targeted degradation of aggregation-prone proteins and protein aggregates (e.g., huntingtin, TDP-43, and FUS mutants), as well as organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum). These antibody-fusion-based AceTAC degraders were designed to mimic the function of autophagy receptors, simultaneously binding with the cellular targets and the LC3 proteins on the autophagosomal membrane, eventually transporting the target to the autophagy-lysosomal process for degradation. The AceTAC degradation system provides design principles for antibody-based degradation through autophagy, largely expanding the scope of intracellular targeted degradation technologies.

Autophagy in Parkinson's Disease.

Parkinson's disease (PD) is a devastating disease associated with accumulation of α-synuclein (α-Syn) within dopaminergic neurons, leading to neuronal death. PD is characterized by both motor and non-motor clinical symptoms. Several studies indicate that autophagy, an important intracellular degradation pathway, may be involved in different neurodegenerative diseases including PD. The autophagic process mediates the degradation of protein aggregates, damaged and unneeded proteins, and organelles, allowing their clearance, and thereby maintaining cell homeostasis. Impaired autophagy may cause the accumulation of abnormal proteins. Incomplete or impaired autophagy may explain the neurotoxic accumulation of protein aggregates in several neurodegenerative diseases including PD. Indeed, studies have suggested the contribution of impaired autophagy to α-Syn accumulation, the death of dopaminergic neurons, and neuroinflammation. In this review, we summarize the recent literature on the involvement of autophagy in PD pathogenesis.

Tethering ATG16L1 or LC3 induces targeted autophagic degradation of protein aggregates and mitochondria

ABSTRACT Proteolysis-targeting chimeras (PROTACs) based on the ubiquitin-proteasome system have made great progress in the field of drug discovery. There is mounting evidence that the accumulation of aggregation-prone proteins or malfunctioning organelles is associated with the occurrence of various age-related neurodegenerative disorders and cancers. However, PROTACs are inefficient for the degradation of such large targets due to the narrow entrance channel of the proteasome. Macroautophagy (hereafter referred to as autophagy) is known as a self-degradative process involved in the degradation of bulk cytoplasmic components or specific cargoes that are sequestered into autophagosomes. In the present study, we report the development of a generalizable strategy for the targeted degradation of large targets. Our results suggested that tethering large target models to phagophore-associated ATG16L1 or LC3 induced targeted autophagic degradation of the large target models. Furthermore, we successfully applied this autophagy-targeting degradation strategy to the targeted degradation of HTT65Q aggregates and mitochondria. Specifically, chimeras consisting of polyQ-binding peptide 1 (QBP) and ATG16L1-binding peptide (ABP) or LC3-interacting region (LIR) induced targeted autophagic degradation of pathogenic HTT65Q aggregates; and the chimeras consisting of mitochondria-targeting sequence (MTS) and ABP or LIR promoted targeted autophagic degradation of dysfunctional mitochondria, hence ameliorating mitochondrial dysfunction in a Parkinson disease cell model and protecting cells from apoptosis induced by the mitochondrial stress agent FCCP. Therefore, this study provides a new strategy for the selective proteolysis of large targets and enrich the toolkit for autophagy-targeting degradation.

Asteroid bodies and autophagy.

Asteroid bodies are star-shaped inclusions in the cytoplasm of multinucleated giant cells. Asteroid bodies are not pathognomonic for a single disease, as these structures may be encountered in various granulomatous inflammatory conditions, including sarcoidosis, foreign body-type granulomas, and mycotic granulomas. In hematoxylin and eosin-stained light-microscopic preparations, asteroid bodies are strongly eosinophilic and surrounded by a halo of small clear vacuoles, as shown in Figures 1-3. Their bright red color reflects a high protein content, whereas the halo of clear vacuoles likely represents an artifact due to dissolved lipid material. In the past, asteroid bodies have intrigued several researchers and electron microscopy has been applied to unravel the composition and pathogenesis of this most peculiar microscopic phenomenon. About 40 years ago, it was discovered that the shape of asteroid bodies in Langhans-type giant cells is determined by the structure of the cytosphere, an ordered central subcellular structure consisting of a pool of centrioles, radiating microtubules, and radially arranged Golgi networks.1 In the 1990s, other investigators observed that the halo of clear vacuoles around the asteroid body contains loosely arranged myelin membranes, possibly representing excessive remnants of cellular membranes formed after the fusion of activated histiocytes.2 Notably, these seminal electron microscopic studies of asteroid bodies may point towards a possible role of autophagy in the formation of asteroid bodies. Herein, it is hypothesized that these structures contain aggregates of misfolded proteins and damaged organelles transported along a microtubular network and delivered to lysosomes in an organizing center close to the Golgi complex. In this pilot immunohistochemical study, evidence is provided that asteroid bodies contain both the autophagy receptor p62 (also known as SQSTM1) and the autophagosome marker LC3. For technical details of immunohistochemistry (IHC) please see the study by Te Rijdt et al.3 At the molecular level, the interaction of LC3 with p62 facilitates the fusion of autophagosomes with lysosomes during the degradation of protein aggregates or cell organelles. Notably, LC3-positive granules become larger in cells where autophagosome formation is abrogated.4 The first case concerns an annular elastolytic granuloma, a rare granulomatous skin disease of unknown etiology and pathogenesis, in which degraded elastic fibers are phagocytized by histiocytes and multinucleated giant cells. In this case, easily discerned asteroid bodies (Figure 1A) contained both p62 (Figure 1B) and granular deposits of LC3. The second case represents a foreign body-type granulomatous disease, in which granulomas had formed around abundant silicone material that had leaked from an elbow prosthesis into surrounding soft tissue (particle disease). In this case, many asteroid bodies were seen in the cytoplasm of multinucleated giant cells that had ingested the silicone rubber (Figure 2A). Here, asteroid bodies also contained both p62 and particularly large granules with LC3 (Figure 2B). Third, Figure 3 represents two different cases of cutaneous sarcoidosis, a disease in which Langhans-type giant cells with characteristic asteroid bodies usually are rare (Figure 3A). As in the other granulomatous inflammatory disorders presented herein, the asteroid body was surrounded by LC3-positive material (Figure 3B). In conclusion, ultrastructural studies published many decades ago have shown that the stellate shape of asteroid bodies is determined by the radial arrangement of a microtubular network around the so-called cytosphere. In this pilot study, IHC staining for the autophagy markers LC3 and p62 indicated that autophagy may play a role in the morphological appearance of asteroid bodies. For instance, it is conceivable that the dynamics of autophagy contribute to the bright red color of aggregated proteins around asteroid cores in hematoxylin and eosin-stained light-microscopic preparations. Moreover, given the remarkably large size of LC3-containing granules found in foreign body-type granulomas, it seems likely that autophagy may become impaired during a rapid cellular turnover when mononuclear histiocytes fuse into multinucleated giant cells. I have no problem concerning data sharing, including the complete text and all three figures.

Curcumin Modulates p62-Keap1-Nrf2-Mediated Autophagy in Rotenone-Induced Parkinson's Disease Mouse Models.

Autophagy mediates self-digestion of abnormally aggregated proteins and organelles present in the cytoplasm. This mechanism may prove to be neuroprotective against Parkinson's disease (PD) by clearing misfolded α-synuclein (α-syn) aggregates from dopaminergic neurons. p62, an adaptor protein acts as a selective substrate for autophagy and regulates the formation as well as the degradation of protein aggregates. p62 sequesters keap1 freeing Nrf2 and consequently activating the transcription of its target genes. In the present study, we aimed to investigate the anti-parkinsonian activity of curcumin targeting primarily activation of autophagy via the Nrf2-Keap1 pathway. The mice were subcutaneously injected with rotenone (2.5 mg/kg bodyweight) and co-treated with oral administration of curcumin (80 mg/kg bodyweight) for 35 days. Following completion of dosing, motor activities, anti-oxidative potential, mitochondrial dysfunction, and various protein expressions, including Nrf2, Keap1, p62, LC3, Bcl2, Bax, and caspase 3, were assessed. The results revealed that curcumin restored the motor coordination and anti-oxidative activity while improving the mitochondrial functioning in PD mice. Autophagy was evaluated by the change in the expression of autophagic markers, p62 and LC3-II. Reduced p62 and LC3-II expressions in the rotenone mouse model of PD confirmed the compromised autophagy pathway, consequently increasing the aggregation of misfolded protein α-syn. Whereas, curcumin treatment-enhanced autophagy-mediated clearance of misfolded α-syn proteins by increasing the LC3-II expression and blocked apoptotic cascade. Curcumin administration upregulated the Nrf2 expression and normalized the Nrf2-Keap1 pathway, which justifies the improved anti-oxidative activity. Therefore, the findings reveal that curcumin is a Nrf2-inducer and is endowed with neuroprotective potential, which may prove to be a potential candidate for the anti-Parkinson's disease treatment therapy.

Enhancement of lysosome biogenesis as a potential therapeutic approach for neurodegenerative diseases.

Millions of people are suffering from Alzheimer's disease globally, but there is still no effective treatment for this neurodegenerative disease. Thus, novel therapeutic approaches for Alzheimer's disease are needed, which requires further evaluation of the regulatory mechanisms of protein aggregate degradation. Lysosomes are crucial degradative organelles that maintain cellular homeostasis. Transcription factor EB-mediated lysosome biogenesis enhances autolysosome-dependent degradation, which subsequently alleviates neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. In this review, we start by describing the key features of lysosomes, including their roles in nutrient sensing and degradation, and their functional impairments in different neurodegenerative diseases. We also explain the mechanisms - especially the post-translational modifications - which impact transcription factor EB and regulate lysosome biogenesis. Next, we discuss strategies for promoting the degradation of toxic protein aggregates. We describe Proteolysis-Targeting Chimera and related technologies for the targeted degradation of specific proteins. We also introduce a group of LYsosome-Enhancing Compounds, which promote transcription factor EB-mediated lysosome biogenesis and improve learning, memory, and cognitive function in APP-PSEN1 mice. In summary, this review highlights the key aspects of lysosome biology, the mechanisms of transcription factor EB activation and lysosome biogenesis, and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.

Novel Insight into Functions of Transcription Factor EB (TFEB) in Alzheimer's Disease and Parkinson's Disease.

A key pathological feature of neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) is the accumulation of aggregated and misfolded protein aggregates with limited effective therapeutic agents. TFEB (transcription factor EB), a key regulator of lysosomal biogenesis and autophagy, plays a pivotal role in the degradation of protein aggregates and has thus been regarded as a promising therapeutic target for these NDs. Here, we systematically summarize the molecular mechanisms and function of TFEB regulation. We then discuss the roles of TFEB and autophagy-lysosome pathways in major neurodegenerative diseases including AD and PD. Finally, we illustrate small molecule TFEB activators with protective roles in NDs animal models, which show great potential for being further developed into novel anti-neurodegenerative agents. Overall, targeting TFEB for enhancing lysosomal biogenesis and autophagy may represent a promising opportunity for the discovery of disease-modifying therapeutics for neurodegenerative disorders though more in-depth basic and clinical studies are required in the future.

Neocryptotanshinone protects against myocardial ischemia-reperfusion injury by promoting autolysosome degradation of protein aggregates via the ERK1/2-Nrf2-LAMP2 pathway

BackgroundAggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein aggregates. However, there have been few mechanism research related to aggrephagy in myocardial ischemia/reperfusion (I/R) injury. Neocryptotanshinone (NCTS) is a fat-soluble active compound extracted from Salvia miltiorrhiza, and may be cardioprotective against I/R. However, the efficacy and specific mechanism of NCTS on I/R have not been studied. PurposeThe current study aimed to investigate the molecular mechanism of NCTS involved in the therapeutic effect on I/R, with a special emphasis on the up-regulation of the ERK1/2-Nrf2-LAMP2 pathway to increase autolysosomal degradation during aggrephagy. MethodsA rat model of myocardial I/R injury was constructed by left anterior descending (LAD) ligation-reperfusion. To verify cardiac protection, autolysosome clearance of protein aggregates, and their intracellular biological mechanism, an oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocyte model was created. ResultsNCTS was found to have a significant cardioprotective effect in I/R rats as evidenced by remarkably improved pathological anatomy, decreased myocardial damage indicators, and substantially enhanced cardiac performance. Mechanistically, NCTS might boost the levels of LAMP2 mRNA and protein, total and Ser40 phosphorylated Nrf2, and Thr202/Tyr204p-ERK1/2 protein. Simultaneously, the cytoplasmic Nrf2 level was reduced after NCTS administration, which was contrary to the total Nrf2 content. However, these beneficial changes were reversed by the co-administration with ERK1/2 inhibitor, PD98059. NCTS therapy up-regulated Rab7 protein content, Cathepsin B activity, and lysosomal acidity, while down-regulating autophagosome numbers, Ubiquitin (Ub), and autophagosome marker protein accumulations through the above signaling pathway. This might indicate that NCTS enhanced lysosomal fusion and hydrolytic capacity. It was also found that NCTS intervention limited oxidative stress and cellular apoptosis both in vivo and in vitro. ConclusionsWe reported for the first time that NCTS promoted the autolysosome removal of protein aggregation both in vivo and in vitro, to exert the therapeutic advantages of myocardial I/R injury. This was reliant on the up-regulation of the ERK1/2-Nrf2-LAMP2 signaling pathway.

NBR1: The archetypal selective autophagy receptor.

NBR1 was discovered as an autophagy receptor not long after the first described vertebrate autophagy receptor p62/SQSTM1. Since then, p62 has currently been mentioned in >10,000 papers on PubMed, while NBR1 is mentioned in <350 papers. Nonetheless, evolutionary analysis reveals that NBR1, and likely also selective autophagy, was present already in the last eukaryotic common ancestor (LECA), while p62 appears first in the early Metazoan lineage. Furthermore, yeast-selective autophagy receptors Atg19 and Atg34 represent NBR1 homologs. NBR1 is the main autophagy receptor in plants that do not contain p62, while most animal taxa contain both NBR1 and p62. Mechanistic studies are starting to shed light on the collaboration between mammalian NBR1 and p62 in the autophagic degradation of protein aggregates (aggrephagy). Several domains of NBR1 are involved in cargo recognition, and the list of known substrates for NBR1-mediated selective autophagy is increasing. Lastly, roles of NBR1 in human diseases such as proteinopathies and cancer are emerging.

The regulatory mechanism and therapeutic potential of transcription factor EB in neurodegenerative diseases.

The autophagy-lysosomal pathway (ALP) is involved in the degradation of protein aggregates and damaged organelles. Transcription factor EB (TFEB), a major regulator of ALP, has emerged as a leading factor in addressing neurodegenerative disease pathology, including Alzheimer's disease (AD), Parkinson's disease (PD), PolyQ diseases, and Amyotrophic lateral sclerosis (ALS). In this review, we delineate the regulation of TFEB expression and its functions in ALP. Dysfunctions of TFEB and its role in the pathogenesis of several neurodegenerative diseases are reviewed. We summarize the protective effects and molecular mechanisms of some TFEB-targeted agonists in neurodegenerative diseases. We also offer our perspective on analyzing the pros and cons of these agonists in the treatment of neurodegenerative diseases from the perspective of drug development. More studies on the regulatory mechanisms of TFEB in other biological processes will aid our understanding of the application of TFEB-targeted therapy in neurodegeneration.

Abstract P2039: The Absence Of Sigmar1 Causes Increased Protein Aggregation And Aggravated Cardiac Dysfunction In R120g- αb-Crystallin Mouse

Background: αB-Crystallin (CryAB) is a molecular chaperone responsible for protecting intermediate filaments by maintaining the structural integrity of the sarcomere. Mutation in CryAB at Arg120Gly (CryAB R120G ) causes the formation of toxic pre-amyloid oligomers and protein aggregation, contributing to cardiac dysfunction. In the event of proteotoxic insult, the chaperone proteins function to prevent the accumulation of protein aggregates. One of the multitasking chaperone proteins abundantly expressed in the heart and known to have molecular roles in the clearance of misfolded protein aggregates is Sigmar1. However, Sigmar1’s role in cardiac proteotoxicity and protein aggregation remains unknown. Hypothesis: We hypothesize that Sigmar1 plays a protective role in the degradation of protein aggregates in the heart. Here, we investigated the functional role of Sigmar1 in misfolded proteins aggregation-induced pathological cardiac remodeling and heart failure in CryAB R120G mice. Methods and Result: We measured the Sigmar1 expression level in CryAB R120G hearts and found that endogenous Sigmar1 protein and transcript levels are substantially reduced in CryAB R120G hearts. To test the functional role of Sigmar1 in a proteotoxic model of heart failure in vivo , we crossed the Sigmar1 knockout mouse with the CryAB R120G mouse to generate Sigmar1 ablated CryAB R120G mouse (CryAB R120G xSigmar1 -/- ). Echocardiographic analysis showed the age-dependent aggravation of cardiac dysfunction in the CryAB R120G xSigmar1 -/- mice compared to CryAB R120G mice. Masson’s Trichrome staining also showed increased cardiac fibrosis in the CryAB R120G xSigmar1 -/- mice hearts. Sigmar1 ablation in CryAB R120G mice hearts resulted in increased protein aggregate content measurement by immunostaining and aggregate size measured by electron microscopy. Sigmar1 siRNA-knockdown in CryAB R120G expressing cardiomyocytes also led to a dramatic increase in aggregate size and content. Conclusion: Overall, we found that Sigmar1 plays a vital role in the degradation of protein aggregates in the heart. Sigmar1 knockdown in CryAB R120G hearts promoted interstitial fibrosis, aggravated ventricular dysfunction, cardiac hypertrophy, and increased protein aggregates.

Coordinative regulation of ERAD and selective autophagy in plants.

Endoplasmic reticulum-associated degradation (ERAD) plays important roles in plant development, hormone signaling, and plant-environment stress interactions by promoting the clearance of certain proteins or soluble misfolded proteins through the ubiquitin-proteasome system. Selective autophagy is involved in the autophagic degradation of protein aggregates mediated by specific selective autophagy receptors. These two major degradation routes co-operate with each other to relieve the cytotoxicity caused by ER stress. In this review, we analyze ERAD and different types of autophagy, including nonselective macroautophagy and ubiquitin-dependent and ubiquitin-independent selective autophagy in plants, and specifically summarize the selective autophagy receptors characterized in plants. In addition to being a part of selective autophagy, ERAD components also serve as their cargos. Moreover, an ubiquitinated substrate can be delivered to two distinguishable degradation systems, while the underlying determinants remain elusive. These excellent findings suggest an interdependent but complicated relationship between ERAD and selective autophagy. According to this point, we propose several key issues that need to be addressed in the future.

The influence of age, sex, and exercise on autophagy, mitophagy, and lysosome biogenesis in skeletal muscle

BackgroundAging decreases skeletal muscle mass and quality. Maintenance of healthy muscle is regulated by a balance between protein and organellar synthesis and their degradation. The autophagy-lysosome system is responsible for the selective degradation of protein aggregates and organelles, such as mitochondria (i.e., mitophagy). Little data exist on the independent and combined influence of age, biological sex, and exercise on the autophagy system and lysosome biogenesis. The purpose of this study was to characterize sex differences in autophagy and lysosome biogenesis in young and aged muscle and to determine if acute exercise influences these processes.MethodsYoung (4–6 months) and aged (22–24 months) male and female mice were assigned to a sedentary or an acute exercise group. Mitochondrial content, the autophagy-lysosome system, and mitophagy were measured via protein analysis. A TFEB-promoter-construct was utilized to examine Tfeb transcription, and nuclear-cytosolic fractions allowed us to examine TFEB localization in sedentary and exercised muscle with age and sex.ResultsOur results indicate that female mice, both young and old, had more mitochondrial protein than age-matched males. However, mitochondria in the muscle of females had a reduced respiratory capacity. Mitochondrial content was only reduced with age in the male cohort. Young female mice had a greater abundance of autophagy, mitophagy, and lysosome proteins than young males; however, increases were evident with age irrespective of sex. Young sedentary female mice had indices of greater autophagosomal turnover than male counterparts. Exhaustive exercise was able to stimulate autophagic clearance solely in young male mice. Similarly, nuclear TFEB protein was enhanced to a greater extent in young male, compared to young female mice following exercise, but no changes were observed in aged mice. Finally, TFEB-promoter activity was upregulated following exercise in both young and aged muscle.ConclusionsThe present study demonstrates that biological sex influences mitochondrial homeostasis, the autophagy-lysosome system, and mitophagy in skeletal muscle with age. Furthermore, our data suggest that young male mice have a more profound ability to activate these processes with exercise than in the other groups. Ultimately, this may contribute to a greater remodeling of muscle in response to exercise training in males.

The Genetics of Autophagy in Multicellular Organisms.

Autophagy, a lysosome-mediated degradation process evolutionarily conserved from yeast to mammals, is essential for maintaining cellular homeostasis and combating diverse cellular stresses. Autophagy involves de novo synthesis of a double-membrane autophagosome, sequestration of selected cellular contents, and subsequent delivery of sequestrated contents to the vacuole (in yeasts and plants) or to lysosomes (in animal cells) for degradation and recycling. Genetic studies in unicellular and multicellular model organisms have systematically revealed the molecular machinery, regulation, and function of autophagy in physiological settings. I review genetic studies in model organisms-from yeast to worm to fly-that enable us to not only identify autophagy genes, including ATG genes and the metazoan-specific EPG genes, but also uncover variants of autophagy in developmental contexts, novel regulatory mechanisms, and signaling events involved in mediating systemic autophagy response. Genetic analysis also helps us understand the liquid-liquid phase separation and transition that control autophagic degradation of protein aggregates. The emerging role of autophagy in zebrafish tissue regeneration is also discussed.

The Regulation of Basal Autophagic Flux by an ATG13‐ATG101 Complex

Autophagy is an essential cellular process by which cellular debris is collected and broken down in the lysosome. Cancer cells rely on autophagy for survival during chemotherapy treatment and periods of hypoxia within solid tumors. In this project, we investigate the ATG13‐ATG101 protein complex, a sub‐complex of the ULK1 initiatory complex whose regulatory role in autophagy is not completely understood. Our recent data demonstrate that an ULK1‐independent ATG13‐ATG101 complex is essential for the basal autophagic degradation of protein aggregates—a process known as aggrephagy. Furthermore, we found that the ULK1‐independent ATG13‐ATG101 complex traffics to condensates of poly‐ubiquitinated protein aggregates where it colocalizes with the autophagy regulator ATG9A.1 Our current data suggest that this ATG13‐ATG101 complex cooperates with ATG9A in the degradation of these protein aggregates. To further elucidate the function of the ATG13‐ATG101 complex, we have developed a protein‐fragment complementation assay using a novel split TurboID (BioID) system in which complementary halves of TurboID are fused to ATG13 and ATG101. Using this split BioID system, together with quantitative LC‐MS/MS and mutants of ATG13 that cannot bind ULK1, we are identifying interactors that are unique to the ULK1‐independent ATG13‐ATG101 complex. Based on previous data, we hypothesize that the ATG13‐ATG101 sub‐complex participates in ATG9A trafficking pathways and recruitment of autophagy regulators to protein aggregate condensates. Thus, our project characterizes the first proximity interactome for the ATG13‐ATG101 complex.1. Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L., BioID reveals an ATG9A interaction with ATG13‐ATG101 in the degradation of p62/SQSTM1‐ubiquitin clusters. EMBO Rep2021, e51136.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

HYPK coordinates degradation of polyneddylated proteins by autophagy

ABSTRACT Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress. Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; Kd, dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D

Macroautophagy and Mitophagy in Neurodegenerative Disorders: Focus on Therapeutic Interventions

Macroautophagy, a quality control mechanism, is an evolutionarily conserved pathway of lysosomal degradation of protein aggregates, pathogens, and damaged organelles. As part of its vital homeostatic role, macroautophagy deregulation is associated with various human disorders, including neurodegenerative diseases. There are several lines of evidence that associate protein misfolding and mitochondrial dysfunction in the etiology of Alzheimer’s, Parkinson’s, and Huntington’s diseases. Macroautophagy has been implicated in the degradation of different protein aggregates such as Aβ, tau, alpha-synuclein (α-syn), and mutant huntingtin (mHtt) and in the clearance of dysfunctional mitochondria. Taking these into consideration, targeting autophagy might represent an effective therapeutic strategy to eliminate protein aggregates and to improve mitochondrial function in these disorders. The present review describes our current understanding on the role of macroautophagy in neurodegenerative disorders and focuses on possible strategies for its therapeutic modulation.

ALSTaR Cells - Novel Stem-cell-based Therapy for Amyotrophic Lateral Sclerosis: A Research Protocol

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that results in the loss of motor neurons leading to limb paralysis and eventual death. Stem cell transplantation can be used to replenish the atrophied motor neurons and slow the progression of the disease, while the use of biomaterials, genetic engineering, and nanoparticles can reduce the hostility of the microenvironment. Thus, we propose a novel combinatorial therapeutic approach termed Amyotrophic Lateral Sclerosis Therapeutic and Regenerative (ALSTaR) cell treatment. Methods: This unified strategy will employ miRNA-124 and chitosan polyplex biomaterial to subsequently enhance engraftment of neural progenitor cells (NPCs), which will be engineered to overexpress an autophagy-regulatory gene, TFEB, and secrete an autophagy-inducing drug, trehalose. We will bioengineer an organoid model of the ventral column of the spinal cord which will be used for extensive in vitro characterization of ALSTaR cells through single-slice electrophysiology and immunocytochemistry, followed by in vivo work. Using the SOD1G93A mutant mouse model, weekly behavioural assessments of motor recovery will be conducted until the humane endpoint; thereafter extensive immunohistochemical and protein quantification analyses will be performed to determine treatment efficacy. Results: ALSTaR cell treatment should result in motor functional recovery and lifespan elongation through several cellular and molecular mechanisms, including decreased neuroinflammation, upregulation of autophagy, degradation of protein aggregates, enhancement of transplanted cell integration, and regeneration of axons. Discussion: In vitro characterization of ALSTaR cells in bioengineered spinal cord organoids will reveal stable electrophysiological recordings of motor neurons, higher levels of neuronal differentiation markers, and lower levels of inflammation markers in the ALSTaR group compared to the control group or the groups treated with just the biomaterial or just the NPCs, suggesting neuronal recovery. Behavioural assessments in the ALS mouse model will reveal increased motor coordination, neuromuscular strength, and motor activity in the ALSTaR group compared to other groups, suggesting motor functional recovery. In vivo characteriz ation of ALSTaR cells in the ALS mouse model will suggest enhanced stem cell integration and recovery of cellular and molecular processes. Conclusion: With currently no effective treatment for ALS, this novel combinatorial treatment strategy could improve the health-related quality of life of patients suffering from this debilitating disease.