The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation.

In humans, aggregation of polyglutamine repeat (polyQ) proteins causes disorders such as Huntington's disease. Although plants express hundreds of polyQ-containing proteins, no pathologies arising from polyQ aggregation have been reported. To investigate this phenomenon, we expressed an aggregation-prone fragment of human huntingtin (HTT) with an expanded polyQ stretch (Q69) in Arabidopsis thaliana plants. In contrast to animal models, we find that Arabidopsis sp. suppresses Q69 aggregation through chloroplast proteostasis. Inhibition of chloroplast proteostasis diminishes the capacity of plants to prevent cytosolic Q69 aggregation. Moreover, endogenous polyQ-containing proteins also aggregate on chloroplast dysfunction. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Synthetic Arabidopsis SPP prevents polyQ-expanded HTT aggregation in human cells. Likewise, ectopic SPP expression in Caenorhabditis elegans reduces neuronal Q67 aggregation and subsequent neurotoxicity. Our findings suggest that synthetic plant proteins, such as SPP, hold therapeutic potential for polyQ disorders and other age-related diseases involving protein aggregation.

Neuroprotective effects of hepatoma-derived growth factor in models of Huntington's disease.

Huntington's disease (HD) is a movement disorder caused by a mutation in the Huntingtin gene that leads to severe neurodegeneration. Molecular mechanisms of HD are not sufficiently understood, and no cure is currently available. Here, we demonstrate neuroprotective effects of hepatoma-derived growth factor (HDGF) in cellular and mouse HD models. We show that HD-vulnerable neurons in the striatum and cortex express lower levels of HDGF than resistant ones. Moreover, lack of endogenous HDGF exacerbated motor impairments and reduced the life span of R6/2 Huntington's disease mice. AAV-mediated delivery of HDGF into the brain reduced mutant Huntingtin inclusion load, but had no significant effect on motor behavior or life span. Interestingly, both nuclear and cytoplasmic versions of HDGF were efficient in rescuing mutant Huntingtin toxicity in cellular HD models. Moreover, extracellular application of recombinant HDGF improved viability of mutant Huntingtin-expressing primary neurons and reduced mutant Huntingtin aggregation in neural progenitor cells differentiated from human patient-derived induced pluripotent stem cells. Our findings provide new insights into the pathomechanisms of HD and demonstrate neuroprotective potential of HDGF in neurodegeneration.

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes.

Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington's and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

Insights Into the Links Between Proteostasis and Aging From C. elegans.

Protein homeostasis (proteostasis) is maintained by a tightly regulated and interconnected network of biological pathways, preventing the accumulation and aggregation of damaged or misfolded proteins. Thus, the proteostasis network is essential to ensure organism longevity and health, while proteostasis failure contributes to the development of aging and age-related diseases that involve protein aggregation. The model organism Caenorhabditis elegans has proved invaluable for the study of proteostasis in the context of aging, longevity and disease, with a number of pivotal discoveries attributable to the use of this organism. In this review, we discuss prominent findings from C. elegans across the many key aspects of the proteostasis network, within the context of aging and disease. These studies collectively highlight numerous promising therapeutic targets, which may 1 day facilitate the development of interventions to delay aging and prevent age-associated diseases.

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

Differences in patient characteristics and external validity of randomized clinical trials on pain management following total hip and knee arthroplasty: a systematic review.

BACKGROUND: The external validity of randomized controlled trials (RCTs) is critical for the relevance of trial results in a clinical setting. We aimed to assess the external validity of RCTs investigating postoperative pain treatment after total hip and knee arthroplasty (THA and TKA) by comparing patient characteristics in these trials with a clinical cohort. Further, we assessed the use of exclusion criteria of the included RCTs. METHODS: We searched PubMed, Embase, and Cochrane Central Register of Controlled Trials for relevant RCTs up to June 2019. Data on patient characteristics from this research population were compared with an unselected clinical cohort from the Danish Hip and Knee Arthroplasty Registries in the period 2005-2019. Trends in patient characteristics and the use of exclusion criteria were assessed with control charts. RESULTS: In total, 550 RCTs with 48 962 participants were included in the research cohort. The clinical cohort included 101 439 THA patients and 90 505 TKA patients. Patient characteristics (age, body mass index (BMI), American Society of Anesthesiologists (ASA) score and sex distribution) in the research cohort resembled those of the clinical cohort. Age, BMI and ASA scores did not change over time in the research cohort. In the clinical cohort, age increased among both THA and TKA patients, and BMI and ASA scores increased among TKA patients. Most commonly used exclusion criteria in the RCTs were high ASA score (62%), older age (45%), obesity (32%) and chronic opioid use (41%). Exclusion of chronic opioid users and individuals with obesity increased over time. CONCLUSION: Patient characteristics in research trials investigating postoperative pain management after THA and TKA currently resemble those of a clinical cohort. However, individuals in the clinical cohort are getting older, and TKA patients more obese with increasing ASA scores. Concomitantly, RCTs increase the tendency to exclude patients with older age, obesity, chronic pain and/or opioid use. This trending discrepancy can hinder the generalizability of future research results, and therefore increased focus on pragmatic trials resembling real-world conditions are needed. PROSPERO REGISTRATION NUMBER: CRD42019125691.

DNAJB6, a Key Factor in Neuronal Sensitivity to Amyloidogenesis.

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

Author Correction: The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington's disease patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The E3 ubiquitin ligase UBR5 interacts with the H/ACA ribonucleoprotein complex and regulates ribosomal RNA biogenesis in embryonic stem cells.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.

The effect of remote ischaemic preconditioning on myocardial injury in emergency hip fracture surgery (PIXIE trial): phase II randomised clinical trial.

OBJECTIVE: To investigate whether remote ischaemic preconditioning (RIPC) prevents myocardial injury in patients undergoing hip fracture surgery. DESIGN: Phase II, multicentre, randomised, observer blinded, clinical trial. SETTING: Three Danish university hospitals, 2015-17. PARTICIPANTS: 648 patients with cardiovascular risk factors undergoing hip fracture surgery. 286 patients were assigned to RIPC and 287 were assigned to standard practice (control group). INTERVENTION: The RIPC procedure was initiated before surgery with a tourniquet applied to the upper arm and consisted of four cycles of forearm ischaemia for five minutes followed by reperfusion for five minutes. MAIN OUTCOME MEASURES: The original primary outcome was myocardial injury within four days of surgery, defined as a peak plasma cardiac troponin I concentration of 45 ng/L or more caused by ischaemia. The revised primary outcome was myocardial injury within four days of surgery, defined as a peak plasma cardiac troponin I concentration of 45 ng/L or more or high sensitive troponin I greater than 24 ng/L (the primary outcome was changed owing to availability of testing). Secondary outcomes were peak plasma troponin I and total troponin I release during the first four days after surgery (cardiac and high sensitive troponin I), perioperative myocardial infarction, major adverse cardiovascular events, and all cause mortality within 30 days of surgery, length of postoperative stay, and length of stay in the intensive care unit. Several planned secondary outcomes will be reported elsewhere. RESULTS: 573 of the 648 randomised patients were included in the intention-to-treat analysis (mean age 79 (SD 10) years; 399 (70%) women). The primary outcome occurred in 25 of 168 (15%) patients in the RIPC group and 45 of 158 (28%) in the control group (odds ratio 0.44, 95% confidence interval 0.25 to 0.76; P=0.003). The revised primary outcome occurred in 57 of 286 patients (20%) in the RIPC group and 90 of 287 (31%) in the control group (0.55, 0.37 to 0.80; P=0.002). Myocardial infarction occurred in 10 patients (3%) in the RIPC group and 21 patients (7%) in the control group (0.46, 0.21 to 0.99; P=0.04). Statistical power was insufficient to draw firm conclusions on differences between groups for the other clinical secondary outcomes (major adverse cardiovascular events, 30 day all cause mortality, length of postoperative stay, and length of stay in the intensive care unit). CONCLUSIONS: RIPC reduced the risk of myocardial injury and infarction after emergency hip fracture surgery. It cannot be concluded that RIPC overall prevents major adverse cardiovascular events after surgery. The findings support larger scale clinical trials to assess longer term clinical outcomes and mortality. TRIAL REGISTRATION: ClinicalTrials.gov NCT02344797.

Prostaglandin signals from adult germ stem cells delay somatic aging of Caenorhabditis elegans.

A moderate reduction of body temperature can induce a remarkable lifespan extension. Here we examine the link between cold temperature, germ line fitness and organismal longevity. We show that low temperature reduces age-associated exhaustion of germ stem cells (GSCs) in Caenorhabditis elegans, a process modulated by thermosensory neurons. Notably, robust self-renewal of adult GSCs delays reproductive aging and is required for extended lifespan at cold temperatures. These cells release prostaglandin E2 (PGE2) to induce cbs-1 expression in the intestine, increasing somatic production of hydrogen sulfide (H2S), a gaseous signaling molecule that prolongs lifespan. Whereas loss of adult GSCs reduces intestinal cbs-1 expression and cold-induced longevity, application of exogenous PGE2 rescues these phenotypes. Importantly, tissue-specific intestinal overexpression of cbs-1 mimics cold-temperature conditions and extends longevity even at warm temperatures. Thus, our results indicate that GSCs communicate with somatic tissues to coordinate extended reproductive capacity with longevity.

The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington's disease patients.

Induced pluripotent stem cells (iPSCs) undergo unlimited self-renewal while maintaining their potential to differentiate into post-mitotic cells with an intact proteome. As such, iPSCs suppress the aggregation of polyQ-expanded huntingtin (HTT), the mutant protein underlying Huntington's disease (HD). Here we show that proteasome activity determines HTT levels, preventing polyQ-expanded aggregation in iPSCs from HD patients (HD-iPSCs). iPSCs exhibit high levels of UBR5, a ubiquitin ligase required for proteasomal degradation of both normal and mutant HTT. Conversely, loss of UBR5 increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs. Moreover, UBR5 knockdown hastens polyQ-expanded aggregation and neurotoxicity in invertebrate models. Notably, UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing polyQ-expanded aggregates in HD-cell models. Besides HTT levels, intrinsic enhanced UBR5 expression determines global proteostasis of iPSCs preventing the aggregation of misfolded proteins ensued from normal metabolism. Thus, our findings indicate UBR5 as a modulator of super-vigilant proteostasis of iPSCs.

Insights into the ubiquitin-proteasome system of human embryonic stem cells.

Human embryonic stem cells (hESCs) exhibit high levels of proteasome activity, an intrinsic characteristic required for their self-renewal, pluripotency and differentiation. However, the mechanisms by which enhanced proteasome activity maintains hESC identity are only partially understood. Besides its essential role for the ability of hESCs to suppress misfolded protein aggregation, we hypothesize that enhanced proteasome activity could also be important to degrade endogenous regulatory factors. Since E3 ubiquitin ligases are responsible for substrate selection, we first define which E3 enzymes are increased in hESCs compared with their differentiated counterparts. Among them, we find HECT-domain E3 ligases such as HERC2 and UBE3A as well as several RING-domain E3s, including UBR7 and RNF181. Systematic characterization of their interactome suggests a link with hESC identity. Moreover, loss of distinct up-regulated E3s triggers significant changes at the transcriptome and proteome level of hESCs. However, these alterations do not dysregulate pluripotency markers and differentiation ability. On the contrary, global proteasome inhibition impairs diverse processes required for hESC identity, including protein synthesis, rRNA maturation, telomere maintenance and glycolytic metabolism. Thus, our data indicate that high proteasome activity is coupled with other determinant biological processes of hESC identity.

Mechanisms of protein homeostasis (proteostasis) maintain stem cell identity in mammalian pluripotent stem cells.

Protein homeostasis, or proteostasis, is essential for cell function, development, and organismal viability. The composition of the proteome is adjusted to the specific requirements of a particular cell type and status. Moreover, multiple metabolic and environmental conditions challenge the integrity of the proteome. To maintain the quality of the proteome, the proteostasis network monitors proteins from their synthesis through their degradation. Whereas somatic stem cells lose their ability to maintain proteostasis with age, immortal pluripotent stem cells exhibit a stringent proteostasis network associated with their biological function and intrinsic characteristics. Moreover, growing evidence indicates that enhanced proteostasis mechanisms play a central role in immortality and cell fate decisions of pluripotent stem cells. Here, we will review new insights into the melding fields of proteostasis and pluripotency and their implications for the understanding of organismal development and survival.

Proteostasis of Huntingtin in Health and Disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by motor dysfunction, cognitive deficits and psychosis. HD is caused by mutations in the Huntingtin (HTT) gene, resulting in the expansion of polyglutamine (polyQ) repeats in the HTT protein. Mutant HTT is prone to aggregation, and the accumulation of polyQ-expanded fibrils as well as intermediate oligomers formed during the aggregation process contribute to neurodegeneration. Distinct protein homeostasis (proteostasis) nodes such as chaperone-mediated folding and proteolytic systems regulate the aggregation and degradation of HTT. Moreover, polyQ-expanded HTT fibrils and oligomers can lead to a global collapse in neuronal proteostasis, a process that contributes to neurodegeneration. The ability to maintain proteostasis of HTT declines during the aging process. Conversely, mechanisms that preserve proteostasis delay the onset of HD. Here we will review the link between proteostasis, aging and HD-related changes.

Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan.

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.