Mitochondrial dysfunction and impaired mitophagy are hallmarks of ageing and age-related pathologies. Disrupted inter-organellar communication among mitochondria, endoplasmic reticulum (ER), and lysosomes, further contributes to cellular dysfunction. While mitophagy has emerged as a promising target for neuroprotection and geroprotection, its potential to restore age-associated defects in organellar crosstalk remains unclear. Here, we show that mitophagy deficiency deregulates the morphology and homeostasis of mitochondria, ER and lysosomes, mirroring age-related alterations. In contrast, Urolithin A (UA), a gut-derived metabolite and potent mitophagy inducer, restores inter-organellar communication via calcium signaling, thereby, promoting mitophagy, healthspan and longevity. Our multi-omic analysis reveals that UA reorganizes ER, mitochondrial and lysosomal networks, linking inter-organellar dynamics to mitochondrial quality control. In Caenorhabditis elegans, UA induces calcium release from the ER, enhances lysosomal activity, and drives DRP-1/DNM1L/DRP1-mediated mitochondrial fission, culminating in efficient mitophagy. Calcium chelation abolishes UA-induced mitophagy, blocking its beneficial impact on muscle function and lifespan, underscoring the critical role of calcium signaling in UA's geroprotective effects. Furthermore, UA-induced calcium elevation activates mitochondrial biogenesis via UNC-43/CAMK2D and SKN-1/NFE2L2/Nrf2 pathways, which are both essential for healthspan and lifespan extension. Similarly, in mammalian cells, UA increases intracellular calcium, enhances mitophagy and mitochondrial metabolism, and mitigates stress-induced senescence in a calcium-dependent manner. Our findings uncover a conserved mechanism by which UA-induced mitophagy restores inter-organellar communication, supporting cellular homeostasis and organismal health.Abbreviations: Ca(2+): calcium ions; BJ: human foreskin fibroblasts; BNIP3: BCL2 interacting protein 3; BP: bipyridyl; CAMK2D: calcium/calmodulin dependent protein kinase II delta; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DEGs: differentially expressed genes; DEPs : differentially expressed peptides; DFP: deferiprone; DNM1L/DRP1: dynamin 1 like; EGTA: ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; EMC: endoplasmic reticulum membrane protein complex; ER: endoplasmic reticulum; FCCP: carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; GO: gene ontology; GSVA: Gene Set Variation Analysis; HUVECs: human umbilical vein endothelial cells; IMM: inner mitochondrial membrane; ITPR/InsP3R: inositol 1,4,5-triphosphate receptor; MAM: mitochondria-associated ER membrane; MAPK: mitogen-activated protein kinase; MCU: mitochondrial calcium uniporter; MEFs: mouse embryonic fibroblasts; NAC : N-acetylcysteine; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; NMN: nicotinamide mononucleotide; NR: nicotinamide riboside; OMM: outer mitochondrial membrane; PCA: principal-component analysis; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PQ: paraquat; TMCO: transmembrane and coiled-coil domains 1; TMRE: tetramethylrhodamine ethyl ester perchlorate; UA: urolithin A; VDAC: voltage dependent anion channel.

Mitochondrial dysfunction is a shared hallmark of neurodegenerative disorders, including Alzheimer's disease (AD) and tauopathies among others. Pathological alterations of the microtubule-associated protein Tau can disrupt mitochondrial dynamics, transport, and function, ultimately leading to neuronal toxicity and synaptic deficits. Understanding these processes is crucial for developing therapeutic interventions. The nematode Caenorhabditis elegans serves as a powerful model to study mitochondrial morphology and Tau-induced neurotoxicity due to its well-characterized nervous system and genetic tractability. Here, we describe a robust methodology for assessing mitochondrial morphology, Tau aggregation, and neuronal integrity in a nematode model of tauopathy. By combining confocal laser scanning microscopy and motility assays, we provide a comprehensive framework for investigating mitochondrial deficits. This approach offers valuable insights into the interplay between Tau pathology and mitochondrial dysfunction, thereby advancing our understanding of neurodegenerative mechanisms and potential therapeutic targets.

As global life expectancy continues to rise, the need to understand and mitigate the effects of aging on human physiology has become increasingly important. Aging is characterized by cellular and functional decline, resulting in a higher prevalence of chronic diseases. Model organisms, such as Caenorhabditis elegans, provide valuable insights into the molecular mechanisms underlying aging and serve as platforms for developing potential therapeutic interventions. This chapter highlights the utility of C. elegans in aging research by presenting three straightforward protocols: the lifespan assay, thrashing assay, and lipofuscin accumulation assay. These assays are designed to effectively assess key physiological aspects of organismal health and provide a reliable framework for monitoring the aging process and evaluating anti-aging compounds. Here, we demonstrate the application of these protocols using Urolithin A as an example, underscoring their efficacy in advancing our understanding of aging and contributing to the development of potential interventions.

Calcium signaling plays a pivotal role in diverse cellular processes through precise spatiotemporal regulation and interaction with effector proteins across distinct subcellular compartments. Mitochondria, in particular, act as central hubs for calcium buffering, orchestrating energy production, redox balance and apoptotic signaling, among others. While controlled mitochondrial calcium uptake supports ATP synthesis and metabolic regulation, excessive accumulation can trigger oxidative stress, mitochondrial membrane permeabilization, and cell death. Emerging findings underscore the intricate interplay between calcium homeostasis and mitophagy, a selective type of autophagy for mitochondria elimination. Although the literature is still emerging, this review delves into the bidirectional relationship between calcium signaling and mitophagy pathways, providing compelling mechanistic insights. Furthermore, we discuss how disruptions in calcium homeostasis impair mitophagy, contributing to mitochondrial dysfunction and the pathogenesis of common neurodegenerative diseases.

The Rab3 protein family is composed of a series of small GTP-binding proteins, including Rab3a, Rab3b, Rab3c, and Rab3d, termed Rab3s. They play crucial roles in health, including in brain function, such as through the regulation of synaptic transmission and neuronal activities. In the high-energy-demanding and high-traffic neurons, the Rab3s regulate essential cellular processes, including trafficking of synaptic vesicles and lysosomal positioning, which are pivotal for the maintenance of synaptic integrity and neuronal physiology. Emerging findings suggest that alterations in Rab3s expression are associated with age-related neurodegenerative pathologies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, among others. Here, we provide an overview of how Rab3s dysregulation disrupts neuronal homeostasis, contributing to impaired autophagy, synaptic dysfunction, and eventually leading to neuronal death. We highlight emerging questions on how Rab3s safeguards the brain and how their dysfunction contributes to the different neurodegenerative diseases. We propose fine-tuning the Rab3s signaling directly or indirectly, such as via targeting their upstream protein AMPK, holding therapeutic potential.