Jerome Mertens

Biological aging drives cellular dysfunction and human disease, yet studying human aging dynamics remains challenging due to limited experimental platforms. Here we show that long‐term post‐mitotic culture of human fibroblasts recapitulates and accelerates features of in vivo aging. Longitudinal paired transcriptomic and epigenetic analyses of four female fibroblast lines revealed that in vitro aging reflects key signatures of in vivo primary fibroblast aging. Direct neuronal conversion of in vitro‐aged fibroblasts preserved biological age, enabling pseudo‐longitudinal modeling of neuronal aging through direct reprogramming. Single‐cell transcriptomics of in vitro‐aging fibroblasts revealed a time‐dependent increase in age‐heterogeneity, consistent with the previously reported age‐associated heterogeneity driven by the variable loss of transcriptional programs in vivo. Using this accelerated aging platform, we evaluated anti‐aging compounds: Metformin broadly attenuated transcriptomic and epigenetic aging, while Rapamycin showed limited efficacy. These findings align with pre‐clinical evidence, providing a platform to evaluate and predict therapeutic anti‐aging compound efficacy at molecular resolution. This system advances our understanding of aging mechanisms and facilitates the development of interventions against age‐related diseases.

Summary: Metabolic alterations and dysregulated alternative RNA splicing are central layers of Alzheimer’s disease (AD) pathogenesis, yet whether and to what extent these changes are mechanistically linked in adult human neurons in the aging brain remains unclear. To assess this, we generated induced neurons (iNs) by direct conversion of fibroblasts from sporadic AD patients and age-matched control donors. Fibroblast-derived iNs retaining donor-specific aging signatures have been shown to display several AD-related phenotypes. Metabolomic analysis revealed that AD iNs divert citrate toward ATP-citrate lyase (ACLY)-dependent citrate-to-acetyl-CoA flux, which drives increased nuclear levels of the high-energy metabolite acetyl-CoA. Acetyl-proteomics showed that acetyl-CoA accumulation leads to widespread hyperacetylation of RNA-binding proteins (RBPs) and splice regulators, suggestive of a direct mechanistic link between a metabolic shift and RNA processing. Indeed, single-cell long-read RNA sequencing identifies splicing alterations in AD-relevant transcripts whose isoform usage mirrors changes observed in post-mortem AD cortex samples. Subcellular acetyl-CoA imaging reveals that inhibition of ACLY enzymatic activity selectively reduces nuclear acetyl-CoA levels, and further improves citrate metabolic flux, normalizes RBP acetylation, and re-establishes a normal neuronal splicing landscape in AD iNs. Together, these findings reveal a nuclear acetyl-CoA-RNA processing axis that links age- and disease-related metabolic alterations to alternative splicing dysregulation in AD, and identify neuronal nuclear acetyl-CoA production as a causal and reversible node for restoring metabolic and RNA homeostasis in aging human AD neurons. Highlights: Sporadic Alzheimer’s disease (AD) patient-derived directly induced neurons (iNs) shift metabolism toward nuclear acetyl-CoA production via ATP-citrate lyase (ACLY). Elevated nuclear acetyl-CoA causes hyperacetylation of RNA-binding proteins (RBPs). Patient iNs show pathogenic RNA splicing patterns similar to post-mortem AD brains. Inhibition of ACLY normalizes neuronal acetyl-CoA metabolism and restores RNA splicing homeostasis.

Autophagy is widely proposed to decline with age; however, direct evidence across human cell types remains limited. Moreover, it is unclear whether age‐associated changes in autophagy‐gene transcription are accompanied by corresponding changes in autophagic activity, and whether autophagic activity relates to physiological function during aging. We performed transcriptomic and functional autophagy analyses across subject‐matched human cell types from a healthy aging cohort. Autophagy‐related gene expression increased with age in primary dermal fibroblasts and, to a lesser extent, in induced neurons (iNs). However, autophagy flux was cell type‐ and sex‐specific and uncoupled from transcriptional remodeling. Autophagy flux decreased in male fibroblasts, remained stable in female fibroblasts, and increased in female iNs with age. In freshly isolated peripheral blood mononuclear cells (PBMCs), autophagy flux became increasingly heterogeneous with age and trended higher in older individuals, independent of sex. Associations between autophagy flux and physiological function varied across the adult lifespan; however, in adults aged > 70 years, higher autophagy flux was associated with reduced physical function. In a pilot intervention study, PBMC autophagy flux decreased following 12 weeks of mild exercise in parallel with improved physical function, suggesting that autophagic activity in PBMCs is responsive to physiological intervention in late life. Together, these findings show that autophagy is remodeled in a cell type‐, sex‐ and physiological function‐dependent manner during aging, challenge the view that autophagy uniformly declines with age, and suggest that elevated autophagy flux in older adults may reflect compensatory responses to age‐associated stress rather than enhanced autophagic capacity.

We explored the epigenomic effects of vegan diet (VD) versus meat‐rich diet (MR) and identified mechanisms that help to explain how VD affects epigenetic gene regulation. Genome‐wide DNA methylation profiles in 48 healthy individuals were investigated after a 1‐month randomized isocaloric dietary intervention comparing effects of a VD versus a MR. Genome‐wide DNA methylation analysis revealed changes in differentially methylated positions following dietary intervention, with the VD group showing a higher degree of gene‐promoter silencing in cancer‐related pathways and cell growth‐associated pathways (mTOR and Hippo). Cell type deconvolution indicated an anti‐inflammatory shift in the VD group, characterized by decreased neutrophils and increased CD4 ⁺ T‐cell proportions, aligning with whole blood count data. Pathway analysis reflected changes in gene promoter methylation consistent with macronutrient changes according to the diets, in that VD silenced lipid metabolism and increased insulin pathway genes. Interestingly, the health‐outcome‐trained epigenetic clocks PhenoAge and GrimAge indicated a deceleration of biological aging in the VD group, while the chronologically optimized Blood&Skin clock showed an opposing acceleration. Convergent evidence from pathway analysis and health‐outcome epigenetic clocks thus suggests that a 1‐month VD drives epigenomic changes associated with reduced cancer risk and decelerated biological aging.

Autophagy is widely proposed to decline with age; however, direct evidence for this across cell and tissue types in humans remains limited. Furthermore, it remains unknown whether interventions that improve physiological health during aging can modify autophagic activity in humans. Here, we performed transcriptomic and functional autophagy analyses across subject-matched human cell types from a healthy aging cohort spanning the adult lifespan. RNA-seq of primary dermal fibroblasts and induced neurons (iNs) revealed increased transcription of many autophagy-related genes with age, most markedly in fibroblasts. The impact of age on autophagic activity, measured using autophagy flux assays, was cell type- and sex-dependent, and uncoupled from autophagy-gene transcription. Autophagy flux decreased with age in male fibroblasts, was unchanged in female fibroblasts, and increased in female iNs. In freshly isolated peripheral blood mononuclear cells (PBMCs), autophagy flux became more heterogeneous with age and trended higher in older individuals, independent of sex. Although autophagy flux levels did not match across different cell types, higher autophagy flux in all cell types was associated with reduced physical function in older adults (≥70 years). Importantly, autophagy flux decreased following 12 weeks of mild exercise in parallel with improved physical function. These findings indicate that autophagy is regulated in a cell type-, sex-and physiological function-dependent manner during human aging, and highlight PBMC autophagy flux as a potentially modifiable, blood-accessible readout of physiological state in older adults.

SUMMARY: While Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for late-onset Alzheimer’s disease, its mechanistic role in the brain-resident macrophage, microglia, remains elusive. Microglia are important in the clearance of pathology in disease, heavily relying on lysosome functionality; therefore, we sought to understand the impact of APOE4 on microglial function. APOE44 microglia have been shown to have lipid accumulation, yet the mechanisms leading to this accumulation are unknown. Using induced pluripotent stem cell-derived microglia, we found that the APOE4 haplotype resulted in transcriptional state shifts in microglia, suppressing activated-response microglia (ARMs) and promoting a G2 senescent-like state. We found that APOE44 microglia accumulate cholesterol esters and provide less lipid support to fibroblast-induced neurons, decreasing their synaptic connections. APOE44 microglia secrete significantly less lipoproteins, leading to the accumulation of lipoproteins within the cells including the lysosomes. APOE44 microglia exhibit impaired lysosomal acidification and degradation capacity. Further, our results elucidated that APOE44 microglia are proinflammatory and shift away from fatty acid oxidation towards glycolysis, due to dysfunctional mitochondria. Taken together, our findings indicate that a loss-of-function in lipoprotein secretion drives intracellular lipid accumulation, including within lysosomes, ultimately disrupting the lysosome-endoplasmic reticulum-mitochondrial axis. This drives a proinflammatory and metabolically compromised microglial phenotype with impaired neuro-supportive functions. GRAPHICAL

The design of human model systems is highly relevant to unveil the underlying mechanisms of aging and to provide insights on potential interventions to extend human health and life span. In this perspective, we explore the potential of 2D or 3D culture models comprising human induced pluripotent stem cells and transdifferentiated cells obtained from aged or age-related disorder-affected donors to enhance our understanding of human aging and to catalyze the discovery of anti-aging interventions.