Mitochondria at the crossroads of health and disease.

Mitochondria reside at the crossroads of catabolic and anabolic metabolism-the essence of life. How their structure and function are dynamically tuned in response to tissue-specific needs for energy, growth repair, and renewal is being increasingly understood. Mitochondria respond to intrinsic and extrinsic stresses and can alter cell and organismal function by inducing metabolic signaling within cells and to distal cells and tissues. Here, we review how the centrality of mitochondrial functions manifests in health and a broad spectrum of diseases and aging.

Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo.

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

Cytoplasmic DNA: sources, sensing, and role in aging and disease.

Endogenous cytoplasmic DNA (cytoDNA) species are emerging as key mediators of inflammation in diverse physiological and pathological contexts. Although the role of endogenous cytoDNA in innate immune activation is well established, the cytoDNA species themselves are often poorly characterized and difficult to distinguish, and their mechanisms of formation, scope of function and contribution to disease are incompletely understood. Here, we summarize current knowledge in this rapidly progressing field with emphases on similarities and differences between distinct cytoDNAs, their underlying molecular mechanisms of formation and function, interactions between cytoDNA pathways, and therapeutic opportunities in the treatment of age-associated diseases.

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

A role of PIEZO1 in iron metabolism in mice and humans.

Iron overload causes progressive organ damage and is associated with arthritis, liver damage, and heart failure. Elevated iron levels are present in 1%-5% of individuals; however, iron overload is undermonitored and underdiagnosed. Genetic factors affecting iron homeostasis are emerging. Individuals with hereditary xerocytosis, a rare disorder with gain-of-function (GOF) mutations in mechanosensitive PIEZO1 ion channel, develop age-onset iron overload. We show that constitutive or macrophage expression of a GOF Piezo1 allele in mice disrupts levels of the iron regulator hepcidin and causes iron overload. We further show that PIEZO1 is a key regulator of macrophage phagocytic activity and subsequent erythrocyte turnover. Strikingly, we find that E756del, a mild GOF PIEZO1 allele present in one-third of individuals of African descent, is strongly associated with increased plasma iron. Our study links macrophage mechanotransduction to iron metabolism and identifies a genetic risk factor for increased iron levels in African Americans.

Mitochondrial Proteases: Multifaceted Regulators of Mitochondrial Plasticity.

Mitochondria are essential metabolic hubs that dynamically adapt to physiological demands. More than 40 proteases residing in different compartments of mitochondria, termed mitoproteases, preserve mitochondrial proteostasis and are emerging as central regulators of mitochondrial plasticity. These multifaceted enzymes limit the accumulation of short-lived, regulatory proteins within mitochondria, modulate the activity of mitochondrial proteins by protein processing, and mediate the degradation of damaged proteins. Various signaling cascades coordinate the activity of mitoproteases to preserve mitochondrial homeostasis and ensure cell survival. Loss of mitoproteases severely impairs the functional integrity of mitochondria, is associated with aging, and causes pleiotropic diseases. Understanding the dual function of mitoproteases as regulatory and quality control enzymes will help unravel the role of mitochondrial plasticity in aging and disease.

Caloric Restriction Reprograms the Single-Cell Transcriptional Landscape of Rattus Norvegicus Aging.

Aging causes a functional decline in tissues throughout the body that may be delayed by caloric restriction (CR). However, the cellular profiles and signatures of aging, as well as those ameliorated by CR, remain unclear. Here, we built comprehensive single-cell and single-nucleus transcriptomic atlases across various rat tissues undergoing aging and CR. CR attenuated aging-related changes in cell type composition, gene expression, and core transcriptional regulatory networks. Immune cells were increased during aging, and CR favorably reversed the aging-disturbed immune ecosystem. Computational prediction revealed that the abnormal cell-cell communication patterns observed during aging, including the excessive proinflammatory ligand-receptor interplay, were reversed by CR. Our work provides multi-tissue single-cell transcriptional landscapes associated with aging and CR in a mammal, enhances our understanding of the robustness of CR as a geroprotective intervention, and uncovers how metabolic intervention can act upon the immune system to modify the process of aging.

A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.

Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Chromatin and Metabolism.

Chromatin is a mighty consumer of cellular energy generated by metabolism. Metabolic status is efficiently coordinated with transcription and translation, which also feed back to regulate metabolism. Conversely, suppression of energy utilization by chromatin processes may serve to preserve energy resources for cell survival. Most of the reactions involved in chromatin modification require metabolites as their cofactors or coenzymes. Therefore, the metabolic status of the cell can influence the spectra of posttranslational histone modifications and the structure, density and location of nucleosomes, impacting epigenetic processes. Thus, transcription, translation, and DNA/RNA biogenesis adapt to cellular metabolism. In addition to dysfunctions of metabolic enzymes, imbalances between metabolism and chromatin activities trigger metabolic disease and life span alteration. Here, we review the synthesis of the metabolites and the relationships between metabolism and chromatin function. Furthermore, we discuss how the chromatin response feeds back to metabolic regulation in biological processes.

Protein Quality Control Degradation in the Nucleus.

Nuclear proteins participate in diverse cellular processes, many of which are essential for cell survival and viability. To maintain optimal nuclear physiology, the cell employs the ubiquitin-proteasome system to eliminate damaged and misfolded proteins in the nucleus that could otherwise harm the cell. In this review, we highlight the current knowledge about the major ubiquitin-protein ligases involved in protein quality control degradation (PQCD) in the nucleus and how they orchestrate their functions to eliminate misfolded proteins in different nuclear subcompartments. Many human disorders are causally linked to protein misfolding in the nucleus, hence we discuss major concepts that still need to be clarified to better understand the basis of the nuclear misfolded proteins' toxic effects. Additionally, we touch upon potential strategies for manipulating nuclear PQCD pathways to ameliorate diseases associated with protein misfolding and aggregation in the nucleus.

Regulation of RNA Polymerase I Transcription in Development, Disease, and Aging.

Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I-III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.

Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.

The nucleolus as a multiphase liquid condensate.

The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid-liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction.

Protein Misfolding Diseases.

The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

Mechanisms and Functions of Spatial Protein Quality Control.

A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.

mTOR at the nexus of nutrition, growth, ageing and disease.

The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention.

Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor.

The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.

mTOR Signaling in Growth, Metabolism, and Disease.

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.