Gene therapy in Parkinson's disease: targeting the endplasmic reticulum proteostasis network

Parkinson’s disease（PD）is the second most common neurodegenerative disease affecting 1%of the population over 60 years of age.The progressive degeneration of dopaminergic neurons at the substantia nigra pars compacta（SNpc）results in a severe and gradual depletion of dopamine content in the striatum,a phenomena that is responsible for the characteristic motor symptoms of this disease.

Myelinosome organelles in pathological retinas: ubiquitous presence and dual role in ocular proteostasis maintenance

The timely and efficient elimination of aberrant proteins and damaged organelles, formed in response to various genetic and environmental stressors, is a vital need for all cells of the body. Recent lines of evidence point out several non-classical strategies employed by ocular tissues to cope with aberrant constituents generated in the retina and in the retinal pigmented epithelium cells exposed to various stressors. Along with conventional strategies relying upon the intracellular degradation of aberrant constituents through ubiquitin-proteasome and/or lysosome-dependent autophagy proteolysis, two non-conventional mechanisms also contribute to proteostasis maintenance in ocular tissues. An exosome-mediated clearing and a myelinosome-driven secretion mechanism do not require intracellular degradation but provide the export of aberrant constituents and “waste proteins” outside of the cells. The current review is centered on the non-degradative myelinosome-driven secretion mechanism, which operates in the retina of transgenic Huntington’s disease R6/1 model mice. Myelinosome-driven secretion is supported by rare organelles myelinosomes that are detected not only in degenerative Huntington’s disease R6/1 retina but also in various pathological states of the retina and of the retinal pigmented epithelium. The intra-retinal traffic and inter-cellular exchange of myelinosomes was discussed in the context of a dual role of the myelinosome-driven secretion mechanism for proteostasis maintenance in different ocular compartments. Special focus was made on the interplay between degradative and non-degradative strategies in ocular pathophysiology, to delineate potential therapeutic approaches to counteract several vision diseases.

ER exit pathways and the control of proteostasis:Crucial role of the UPR,COPII,and ER-phagy in the secretory pathway

The endoplasmic reticulum(ER)is the site of entry of all proteins that function in the secretory pathway including the extracellular environment.Because it controls the folding of newly synthesized secretory proteins,the ER is indispensable for the maintenance of proteostasis in the secretory pathway.Within the ER and,in part,in post-ER compartments,the quality control of protein folding is under the regulation of the unfolded protein response(UPR)pathways.The UPR strategy is to enhance protein folding,increase the ER degradation pathway of misfolded proteins,and allow the exit from the ER of only correctly folded proteins.The latter is controlled by the multimeric complex COPII,which also provides some of the components for ER-phagy the only route for the disposal of protein aggregates.In this overview,we wish to contribute to the introduction of new perspectives in the study of the mechanisms underlying the control of proteostasis within the secretory pathway.

Chloroplast proteostasis:A story of birth,life,and death

Protein homeostasis(proteostasis)is a dynamic balance of protein synthesis and degradation.Because of the endosymbiotic origin of chloroplasts and the massive transfer of their genetic information to the nucleus of the host cell,many protein complexes in the chloroplasts are constituted from subunits encoded by both genomes.Hence,the proper function of chloroplasts relies on the coordinated expression of chloroplast-and nucleus-encoded genes.The biogenesis and maintenance of chloroplast proteostasis are dependent on synthesis of chloroplast-encoded proteins,import of nucleus-encoded chloroplast proteins from the cytosol,and clearance of damaged or otherwise undesired“old”proteins.This review focuses on the regulation of chloroplast proteostasis,its interactionwith proteostasis of the cytosol,and its retrograde control over nuclear gene expression.We also discuss significant issues and perspectives for future studies and potential applications for improving the photosynthetic performance and stress tolerance of crops.

Molecular targets for modulating the protein translation vital to proteostasis and neuron degeneration in Parkinson’s disease

Parkinson’s disease(PD)is the most common neurodegenerative movement disorder,which is characterized by the progressive loss of dopaminergic neurons in the Substantia Nigra pars compacta concomitant with Lewy body formation in affected brain areas.The detailed pathogenic mechanisms underlying the selective loss of dopaminergic neurons in PD are unclear,and no drugs or treatments have been developed to alleviate progressive dopaminergic neuron degeneration in PD.However,the formation ofα-synuclein-positive protein aggregates in Lewy body has been identified as a common pathological feature of PD,possibly stemming from the consequence of protein misfolding and dysfunctional proteostasis.Proteostasis is the mechanism for maintaining protein homeostasis via modulation of protein translation,enhancement of chaperone capacity and the prompt clearance of misfolded protein by the ubiquitin proteasome system and autophagy.Deregulated protein translation and impaired capacities of chaperone or protein degradation can disturb proteostasis processes,leading to pathological protein aggregation and neurodegeneration in PD.In recent years,multiple molecular targets in the modulation of protein translation vital to proteostasis and dopaminergic neuron degeneration have been identified.The potential pathophysiological and therapeutic significance of these molecular targets to neurodegeneration in PD is highlighted.

Anticarin-β shows a promising antiosteosarcoma effect by specifically inhibiting CCT4 to impair proteostasis

Unlike healthy, non-transformed cells, the proteostasis network of cancer cells is taxed to produce proteins involved in tumor development. Cancer cells have a higher dependency on molecular chaperones to maintain proteostasis. The chaperonin T-complex protein ring complex(TRiC) contains eight paralogous subunits(CCT1-8), and assists the folding of as many as 10% of cytosolic proteome.TRiC is essential for the progression of some cancers, but the roles of TRiC subunits in osteosarcoma remain to be explored. Here, we show that CCT4/TRiC is significantly correlated in human osteosarcoma,and plays a critical role in osteosarcoma cell survival. We identify a compound anticarin-β that can specifically bind to and inhibit CCT4. Anticarin-β shows higher selectivity in cancer cells than in normal cells. Mechanistically, anticarin-β potently impedes CCT4-mediated STAT3 maturation. Anticarin-β displays remarkable antitumor efficacy in orthotopic and patient-derived xenograft models of osteosarcoma.Collectively, our data uncover a key role of CCT4 in osteosarcoma, and propose a promising treatment strategy for osteosarcoma by disrupting CCT4 and proteostasis.

Metabolic and proteostatic differences in quiescent and active neural stem cells

Adult neural stem cells are neurogenesis progenitor cells that play an important role in neurogenesis.Therefore,neural regeneration may be a promising target for treatment of many neurological illnesses.The regenerative capacity of adult neural stem cells can be chara cterized by two states:quiescent and active.Quiescent adult neural stem cells are more stable and guarantee the quantity and quality of the adult neural stem cell pool.Active adult neural stem cells are chara cterized by rapid proliferation and differentiation into neurons which allow for integration into neural circuits.This review focuses on diffe rences between quiescent and active adult neural stem cells in nutrition metabolism and protein homeostasis.Furthermore,we discuss the physiological significance and underlying advantages of these diffe rences.Due to the limited number of adult neural stem cells studies,we refe rred to studies of embryonic adult neural stem cells or non-mammalian adult neural stem cells to evaluate specific mechanisms.

Protein synthesis modulation as a therapeutic approach for amyotrophic lateral sclerosis and frontotemporal dementia

Protein synthesis is essential for cells to perform life metabolic processes.Pathological alterations of protein content can lead to particular diseases.Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding,accumulation,aggregation or mislocalization occur.Some of them(like the unfolded protein response)represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis(also known as proteostasis).This is even more important in neurons,as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age.Several neurodegenerative pathologies such as Alzheimer’s,Parkinson’s,and Huntington’s diseases,amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct,unbalanced protein overload.In amyotrophic lateral sclerosis and frontotemporal dementia,the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa(TDP-43).TDP-43 is an RNA binding protein that participates in RNA metabolism,among other functions.Dysregulation of TDP-43(e.g.aggregation and mislocalization)can dramatically affect neurons,and this has been linked to disease development.Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum.These variants can be causative of degeneration onset and progression.Most neurodegenerative diseases(including amyotrophic lateral sclerosis and frontotemporal dementia)have no cure at the moment;however,modulating translation has recently emerged as an attractive approach that can be performed at several steps(i.e.regulating activation of initiation and elongation factors,inhibiting unfolded protein response activation or inducing chaperone expression and activity).This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis.We strive to highlight the importance of research on drugs that,not only restore protein imbalance without compromising translational activity of cells,but are also as safe as possible for the patients.

CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence

Cullin-RING E3 ubiquitin ligases(CRLs),the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells,represent core cellular machinery for executing protein degradation and maintaining proteostasis.Here,we asked what roles Cullin proteins play in human mesenchymal stem cell(hMSC)homeostasis and senescence.To this end,we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models:replicative senescent hMSCs,Hutchinson-Gilford Progeria Syndrome hMSCs,and Werner syndrome hMSCs.Among all family members,we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence.To investigate CUL2-specific underlying mechanisms,we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells(hESCs).When we differentiated these into h MSCs,we found that CUL2 deletion markedly accelerates hMSC senescence.Importantly,we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2(a known negative regulator of proliferation)through the substrate receptor protein APPBP2,which in turn downregulates one of the canonical aging marker-P21^(waf1/cip1),and thereby delays senescence.Our work provides important insights into how CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts hMSC senescence,providing a molecular basis for directing intervention strategies against aging and aging-related diseases.

Multidimensional autophagy nano-regulator boosts Alzheimer's disease treatment by improving both extra/intraneuronal homeostasis

Intraneuronal dysproteostasis and extraneuronal microenvironmental abnormalities in Alzheimer’s disease(AD)collectively culminate in neuronal deterioration.In the context of AD,autophagy dysfunction,a multi-link obstacle involving autophagy downregulation and lysosome defects in neurons/microglia is highly implicated in intra/extraneuronal pathological processes.Therefore,multidimensional autophagy regulation strategies co-manipulating“autophagy induction”and“lysosome degradation”in dual targets(neuron and microglia)are more reliable for AD treatment.Accordingly,we designed an RP-1 peptide-modified reactive oxygen species(ROS)-responsive micelles(RT-NM)loading rapamycin or gypenoside XVII.Guided by RP-1 peptide,the ligand of receptor for advanced glycation end products(RAGE),RT-NM efficiently targeted neurons and microglia in AD-affected region.This nanocombination therapy activated the whole autophagy-lysosome pathway by autophagy induction(rapamycin)and lysosome improvement(gypenoside XVII),thus enhancing autophagic degradation of neurotoxic aggregates and inflammasomes,and promoting Aβ phagocytosis.Resultantly,it decreased aberrant protein burden,alleviated neuroinflammation,and eventually ameliorated memory defects in 3×Tg-AD transgenic mice.Our research developed a multidimensional autophagy nano-regulator to boost the efficacy of autophagy-centered AD therapy.

Lung Single-Cell Transcriptomics Offers Insights into the Pulmonary Interstitial Toxicity Caused by Silica Nanoparticles

The adverse respiratory outcomes motivated by silica nanoparticles(SiNPs)exposure have received increasing attention.Herein,we aim to elucidate the interplay of diverse cell populations in the lungs and key contributors in triggering lung injuries caused by SiNPs.We conducted a subchronic respiratory exposure model of SiNPs via intratracheal instillation in Wistar rats,where rats were administered with 1.5,3.0,or 6.0 mg/kg body weight SiNPs once a week for 12 times in total.We revealed that SiNPs caused pulmonary interstitial injury in rats by histopatho-logical examination and pulmonary hydroxyproline determination.Further,a single-cell RNA-Seq via screening 10457 cells in the rat lungs disclosed cell-specific responses to SiNPs and cell-to-cell interactions within the alveolar macrophages,epithelial cells,and fibroblasts from rat lungs.These disturbed responses were principally related to the dysregulation of protein homeostasis(proteostasis),accompanied by an inflammatory response in macrophages,cell death in epithelial,proliferation,and extracellular matrix deposition in fibroblast.These cell-specific responses may serve a synergistic role in the pathogenesis of pulmonary interstitial disease triggered by SiNPs.In particular,the analyses of gene interaction networks and gene−disease associations filtered out heat shock proteins(Hsps)family genes crucial to the observed pulmonary lesions caused by SiNPs.Of note,both GEO database analysis and our experiments’validation indicated that Hsps,especially Hspd1,may be a key contributor to pulmonary interstitial injury,possibly through triggering oxidative stress,immune response,and disrupting protein homeostasis.Taken together,our study provides insights into pulmonary toxic effects and underlying molecular mechanisms of SiNPs from a single-cell perspective.

Commentary: PROTACs make undruggable targets druggable: Challenge and opportunity

Proteostasis(protein homeostasis) ensures precise adjustment of cellular demand to proteins in the stress conditions, which is essential in the maintenance of health environment inside cells and is indispensable for the life of organisms1.

Protein misfolding in neurodegenerative diseases:implications and strategies

A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse.Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease,including synthesis,chaperone-assisted folding and trafficking,and degradation via the proteasome and autophagy pathways.Other therapies,like mTOR inhibitors and activators of the heat shock response,can rebalance the entire proteostatic network.However,there are major challenges that impact the development of novel therapies,including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response.A notable development is the creation of collaborative ecosystems that include patients,clinicians,basic and translational researchers,foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent,reverse or delay the progression of neurodegenerative proteinopathies.

Natural compounds in the regulation of proteostatic pathways: An invincible artillery against stress, ageing, and diseases

Cells have different sets of molecules for performing an array of physiological functions.Nucleic acids have stored and carried the information throughout evolution,whereas proteins have been attributed to performing most of the cellular functions.To perform these functions,proteins need to have a unique conformation and a definite lifespan.These attributes are achieved by a highly coordinated protein quality control(PQC)system comprising chaperones to fold the proteins in a proper threedimensional structure,ubiquitin-proteasome system for selective degradation of proteins,and autophagy for bulk clearance of cell debris.Many kinds of stresses and perturbations may lead to the weakening of these protective cellular machinery,leading to the unfolding and aggregation of cellular proteins and the occurrence of numerous pathological conditions.However,modulating the expression and functional efficiency of molecular chaperones,E3 ubiquitin ligases,and autophagic proteins may diminish cellular proteotoxic load and mitigate various pathological effects.Natural medicine and small molecule-based therapies have been well-documented for their effectiveness in modulating these pathways and reestablishing the lost proteostasis inside the cells to combat disease conditions.The present article summarizes various similar reports and highlights the importance of the molecules obtained from natural sources in disease therapeutics.

Chloroplast ROS and stress signaling

Chloroplasts overproduce reactive oxygen species(ROS)under unfavorable environmental conditions,and these ROS are implicated in both signaling and oxidative damage.There is mounting evidence for their roles in translating environmental fluctuations into distinct physiological responses,but their targets,signaling cascades,and mutualism and antagonism with other stress signaling cascades and within ROS signaling remain poorly understood.Great efforts made in recent years have shed new light on chloroplast ROS-directed plant stress responses,from ROS perception to plant responses,in conditional mutants of Arabidopsis thaliana or under various stress conditions.Some articles have also reported the mechanisms underlying the complexity of ROS signaling pathways,with an emphasis on spatiotemporal regulation.ROS and oxidative modification of affected target proteins appear to induce retrograde signaling pathways to maintain chloroplast protein quality control and signaling at a whole-cell level using stress hormones.This review focuses on these seemingly interconnected chloroplast-to-nucleus retrograde signaling pathways initiated by ROS and ROS-modified target molecules.We also discuss future directions in chloroplast stress research to pave the way for discovering new signaling molecules and identifying intersectional signaling components that interact in multiple chloroplast signaling pathways.

Lysosome and proteasome dysfunction in alcohol-induced liver injury

The review describes research findings on the influence of alcohol consumption on two crucial catabolic systems in hepatocytes:the lysosome and the ubiquitin-proteasome system(UPS).The lysosome is a membrane-bound organelle that degrades all aging and/or damaged organelles and hydrolyzes all forms of macromolecules.The UPS is mostly proteolytic.It carries out the majority of its functions in the soluble portion of the cytoplasm(cytosol)and degrades nearly all intracellular proteins,particularly those with short half-lives,so that their levels are tightly controlled.Our review will briefly discuss the epidemiology of alcohol abuse and the spectrum of alcohol-induced liver disease(AILD).We will explain why ethanol(EtOH)metabolism,but not EtOH alone,is hepatotoxic.Then,we will summarize how heavy drinking alters hepatic catabolic systems,resulting in liver enlargement that develops from hepatocyte swelling due,in part,to aberrant accumulation of undegraded lipid droplets(steatosis)and undegraded proteins(proteopathy).Our detailed description of each catabolic system will highlight its discoverer(s)and emphasize each system’s characteristics.Most important,we will review the evidence that chronic EtOH consumption disrupts hepatic lysosome biogenesis and inhibits the UPS by impeding hepatic proteasome activity.It will become evident that each of these EtOH-induced defects has far-reaching functional consequences.Finally,we will describe current and potential therapeutic interventions for alleviating EtOH-induced liver injury.The most effective intervention is the cessation of EtOH consumption.However,there are other potential approaches using natural or synthetic compounds that activate autophagy or the proteasome to enhance the degradation of accumulated lipid droplets or proteins,respectively,which could alleviate AILD.These approaches,now in their early stages of investigation,will also be discussed in this review.

Overcoming drug resistance by targeting protein homeostasis in multiple myeloma

Multiple myeloma(MM)is a plasma cell disorder typically characterized by abundant synthesis of clonal immunoglobulin or free light chains.Although incurable,a deeper understanding of MM pathobiology has fueled major therapeutical advances over the past two decades,significantly improving patient outcomes.Proteasome inhibitors,immunomodulatory drugs,and monoclonal antibodies are among the most effective anti-MM drugs,targeting not only the cancerous cells,but also the bone marrow microenvironment.However,de novo resistance has been reported,and acquired resistance is inevitable for most patients over time,leading to relapsed/refractory disease and poor outcomes.Sustained protein synthesis coupled with impaired/insufficient proteolytic mechanisms makes MM cells exquisitely sensitive to perturbations in protein homeostasis,offering us the opportunity to target this intrinsic vulnerability for therapeutic purposes.This review highlights the scientific rationale for the clinical use of FDA-approved and investigational agents targeting protein homeostasis in MM.

ER stress-induced aggresome trafficking of HtrA1 protects against proteotoxicity

High temperature requirement A1 （HtrA1） belongs to an ancient protein family that is linked to various human disorders. The pre- cise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrAZ remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is impticated in unfotded protein response. ｜n highly secre- tory cells of the retinal pigmented epithelia, endoplasmic reticulum （ER） stress upregulated HtrA1. HtrA1 co-localized with vimen- tin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions dis- played reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mam- malian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.

Interrogating the impact of aggregation-induced emission nanoparticles on in vitro protein stability,ex vivo protein homeostasis,and in vivo biocompatibility

Aggregation-induced emission(AIE)materials offer promising perspectives in disease diagnosis and therapeutics given their unique optical and photochemical properties.A key step toward translational applications for AIE materials is to systematically and vigorously evaluate their biosafety and biocompatibility.While previous studies focus on cellular viability and toxicity,the impact of AIE materials on detailed stress responses manifesting cellular fitness has been less explored.Herein,this work provides the first piece of evidence to support amphiphilic functionalization of AIE nanoparticles minimizes the deterioration on proteome stability and cellular protein homeostasis(proteostasis).To this end,four scaffolds of AIE molecules were prepared,further functionalized into eight nanoparticles with two amphiphilic shells respectively,and characterized for their physicochemical properties.Thermal shift assay quantitatively demonstrates that AIE materials after amphiphilic functionalization into nanoparticles enhance proteome thermodynamic stability and ameliorate proteome aggregation propensity in cellular lysate,echoed by cell viability and fractionation experiments.Intriguingly,poor polydispersity index(PDI)of functionalized nanoparticles exaggerates their retention and aggregation in the cell.Comparative proteomic analysis uncovers that amphiphilic functionalization of AIE materials can minimize the deterioration of cellular protein homeostasis network.Finally,vigorous interrogation of functionalized AIE nanoparticles in mice model reveals the complexity of factors affecting the biocompatibility profiles in vivo,including materials’size,PDI,and treatment frequencies.Overall,amphiphilic functionalization of AIE materials into nanoparticles is necessary to maintain proteome stability and balance cellular protein homeostasis.