Aging induces region-specific dysregulation of hormone synthesis in the primate adrenal gland.

Adrenal glands, vital for steroid secretion and the regulation of metabolism, stress responses and immune activation, experience age-related decline, impacting systemic health. However, the regulatory mechanisms underlying adrenal aging remain largely uninvestigated. Here we established a single-nucleus transcriptomic atlas of both young and aged primate suprarenal glands, identifying lipid metabolism and steroidogenic pathways as core processes impacted by aging. We found dysregulation in centripetal adrenocortical differentiation in aged adrenal tissues and cells in the zona reticularis region, responsible for producing dehydroepiandrosterone sulfate (DHEA-S), were highly susceptible to aging, reflected by senescence, exhaustion and disturbed hormone production. Remarkably, LDLR was downregulated in all cell types of the outer cortex, and its targeted inactivation in human adrenal cells compromised cholesterol uptake and secretion of dehydroepiandrosterone sulfate, as observed in aged primate adrenal glands. Our study provides crucial insights into endocrine physiology, holding therapeutic promise for addressing aging-related adrenal insufficiency and delaying systemic aging.

Intervention with metabolites emulating endogenous cell transitions accelerates muscle regeneration in young and aged mice.

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.

The sirtuin-associated human senescence program converges on the activation of placenta-specific gene PAPPA.

Sirtuins are pro-longevity genes with chromatin modulation potential, but how these properties are connected is not well understood. Here, we generated a panel of isogeneic human stem cell lines with SIRT1-SIRT7 knockouts and found that any sirtuin deficiency leads to accelerated cellular senescence. Through large-scale epigenomic analyses, we show how sirtuin deficiency alters genome organization and that genomic regions sensitive to sirtuin deficiency are preferentially enriched in active enhancers, thereby promoting interactions within topologically associated domains and the formation of de novo enhancer-promoter loops. In all sirtuin-deficient human stem cell lines, we found that chromatin contacts are rewired to promote aberrant activation of the placenta-specific gene PAPPA, which controls the pro-senescence effects associated with sirtuin deficiency and serves as a potential aging biomarker. Based on our survey of the 3D chromatin architecture, we established connections between sirtuins and potential target genes, thereby informing the development of strategies for aging interventions.

Kidney organoid models reveal cilium-autophagy metabolic axis as a therapeutic target for PKD both in vitro and in vivo.

Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

Decoding aging-dependent regenerative decline across tissues at single-cell resolution.

Regeneration across tissues and organs exhibits significant variation throughout the body and undergoes a progressive decline with age. To decode the relationships between aging and regenerative capacity, we conducted a comprehensive single-cell transcriptome analysis of regeneration in eight tissues from young and aged mice. We employed diverse analytical models to study tissue regeneration and unveiled the intricate cellular and molecular mechanisms underlying the attenuated regenerative processes observed in aged tissues. Specifically, we identified compromised stem cell mobility and inadequate angiogenesis as prominent contributors to this age-associated decline in regenerative capacity. Moreover, we discovered a unique subset of Arg1+ macrophages that were activated in young tissues but suppressed in aged regenerating tissues, suggesting their important role in age-related immune response disparities during regeneration. This study provides a comprehensive single-cell resource for identifying potential targets for interventions aimed at enhancing regenerative outcomes in the aging population.

Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response.

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.

Nuclear lamina erosion-induced resurrection of endogenous retroviruses underlies neuronal aging.

The primate frontal lobe (FL) is sensitive to aging-related neurocognitive decline. However, the aging-associated molecular mechanisms remain unclear. Here, using physiologically aged non-human primates (NHPs), we depicted a comprehensive landscape of FL aging with multidimensional profiling encompassing bulk and single-nucleus transcriptomes, quantitative proteome, and DNA methylome. Conjoint analysis across these molecular and neuropathological layers underscores nuclear lamina and heterochromatin erosion, resurrection of endogenous retroviruses (ERVs), activated pro-inflammatory cyclic GMP-AMP synthase (cGAS) signaling, and cellular senescence in post-mitotic neurons of aged NHP and human FL. Using human embryonic stem-cell-derived neurons recapitulating cellular aging in vitro, we verified the loss of B-type lamins inducing resurrection of ERVs as an initiating event of the aging-bound cascade in post-mitotic neurons. Of significance, these aging-related cellular and molecular changes can be alleviated by abacavir, a nucleoside reverse transcriptase inhibitor, either through direct treatment of senescent human neurons in vitro or oral administration to aged mice.

The circadian clock CRY1 regulates pluripotent stem cell identity and somatic cell reprogramming.

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity.

Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells.

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.

Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing.

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

A single-cell transcriptomic atlas of exercise-induced anti-inflammatory and geroprotective effects across the body.

Exercise benefits the whole organism, yet, how tissues across the body orchestrally respond to exercise remains enigmatic. Here, in young and old mice, with or without exercise, and exposed to infectious injury, we characterized the phenotypic and molecular adaptations to a 12-month exercise across 14 tissues/organs at single-cell resolution. Overall, exercise protects tissues from infectious injury, although more effectively in young animals, and benefits aged individuals in terms of inflammaging suppression and tissue rejuvenation, with structural improvement in the central nervous system and systemic vasculature being the most prominent. In vascular endothelial cells, we found that readjusting the rhythmic machinery via the core circadian clock protein BMAL1 delayed senescence and facilitated recovery from infectious damage, recapitulating the beneficial effects of exercise. Our study underscores the effect of exercise in reconstituting the youthful circadian clock network and provides a foundation for further investigating the interplay between exercise, aging, and immune challenges across the whole organism.

A stem cell aging framework, from mechanisms to interventions.

Stem cells play central roles in tissue development, homeostasis, and regeneration. Decades of scientific research have uncovered processes of stem cell decline in tissue and organismal aging, and more recently, pioneering technologies permit the dissection of its underlying mechanisms and inform therapeutic development for aging and aging-associated disorders. In this review, we elucidate aging-related features across different somatic stem cell types, with a specific focus on epigenetic changes, loss of protein homeostasis, and systemic influencing factors, including chronic inflammation, circadian rhythm dysregulation, and metabolic disorder. Our survey of organismal stem cell aging summarizes its underlying biological implications, points to potential biomarkers of stem cell aging, and discusses stem cell-based therapeutic strategies with the potential for promoting healthy aging and combating aging and age-related diseases.

APOE4 drives inflammation in human astrocytes via TAGLN3 repression and NF-κB activation.

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.

LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes.

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.