Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.

Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the study of muscle, mobility and aging (SOMMA).

Autophagy is essential for proteostasis, energetic balance, and cell defense and is a key pathway in aging. Identifying associations between autophagy gene expression patterns in skeletal muscle and physical performance outcomes would further our knowledge of mechanisms related with proteostasis and healthy aging. Muscle biopsies were obtained from participants in the Study of Muscle, Mobility, and Aging (SOMMA). For 575 participants, RNA was sequenced and expression of 281 genes related to autophagy regulation, mitophagy, and mTOR/upstream pathways was determined. Associations between gene expression and outcomes including mitochondrial respiration in muscle fiber bundles (MAX OXPHOS), physical performance (VO2 peak, 400 m walking speed, and leg power), and thigh muscle volume, were determined using negative binomial regression models. For autophagy, key transcriptional regulators including TFE3 and NFKB-related genes (RELA, RELB, and NFKB1) were negatively associated with outcomes. On the contrary, regulators of oxidative metabolism that also promote overall autophagy, mitophagy, and pexophagy (PPARGC1A, PPARA, and EPAS1) were positively associated with multiple outcomes. In line with this, several mitophagy, fusion, and fission-related genes (NIPSNAP2, DNM1L, and OPA1) were also positively associated with outcomes. For mTOR pathway and related genes, expression of WDR59 and WDR24, both subunits of GATOR2 complex (an indirect inhibitor of mTORC1), and PRKAG3, which is a regulatory subunit of AMPK, were negatively correlated with multiple outcomes. Our study identifies autophagy and selective autophagy such as mitophagy gene expression patterns in human skeletal muscle related to physical performance, muscle volume, and mitochondrial function in older persons which may lead to target identification to preserve mobility and independence.

Chaperone-mediated autophagy regulates adipocyte differentiation.

Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst-mediated protein secretion.

Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor ß Signaling.

BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor ß signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor ß signaling and hippocampal function.

Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome.

CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.

Giant tortoise genomes provide insights into longevity and age-related disease.

Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.

Proteostasis alterations in myeloproliferative neoplasms: Oncogenic relevance and therapeutic opportunities.

Myeloproliferative neoplasms (MPNs) represent a frequently occurring group of heterogeneous hematologic malignancies. In the last decade, the identification of JAK2-activating mutations in a significant proportion of MPN patients gave rise to the first molecularly driven therapy for BCR-ABL-negative patients. Nevertheless, current efforts are still focused on the identification of novel therapeutic targets to achieve permanent remission. In this perspective, we focus on the recent findings in this field and highlight new evidence linking proteostasis deregulation with myeloid transformation. We recently reported that the proteostasis regulator AIRAPL acts as a tumor suppressor in MPNs through the modulation of insulin-like growth factor receptor levels at the endoplasmic reticulum. This finding paves the way for new therapeutic approaches to these neoplasms and indicates the importance of protein homeostasis maintenance for normal hematopoiesis.

Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling.

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.