MEC-12/alpha tubulin regulates mitochondrial distribution and mitophagy during oxidative stress in C. elegans.

Mitophagy, the selective removal of dysfunctional mitochondria, is pivotal for the maintenance of neuronal function and survival. MEC-12/α-tubulin contributes to neuronal physiology through the regulation of microtubule assembly, intracellular transport and mitochondrial distribution. However, its role in mitochondrial dynamics and mitophagy remains obscure. Here, we demonstrate that MEC-12 influences mitochondrial morphology under basal conditions and regulates the axonal mitochondrial population. Impairment of MEC-12 results in compromised axonal mitophagy under both basal conditions and oxidative stress. Our results uncover the critical role of MEC-12/α-tubulin for maintaining a healthy mitochondrial population in axons and highlight the complex interplay between microtubules, mitophagy and neuronal health.

Identifying therapeutic compounds for autosomal dominant optic atrophy (ADOA) through screening in the nematode C. elegans.

Autosomal Dominant Optic Atrophy (ADOA) is a rare neurodegenerative condition, characterized by the bilateral loss of vision due to the degeneration of retinal ganglion cells. Its primary cause is linked to mutations in OPA1 gene, which ultimately affect mitochondrial structure and function. The current lack of successful treatments for ADOA emphasizes the need to investigate the mechanisms driving disease pathogenesis and exploit the potential of animal models for preclinical trials. Among such models, Caenorhabditis elegans stands out as a powerful tool, due its simplicity, its genetic tractability, and its relevance to human biology. Despite the lack of a visual system, the presence of mutated OPA1 in the nematode recapitulates ADOA pathology, by stimulating key pathogenic features of the human condition that can be studied in a fast and relatively non-laborious manner. Here, we provide a detailed guide on how to assess the therapeutic efficacy of chemical compounds, in either small or large scale, by evaluating three crucial phenotypes of humanized ADOA model nematodes, that express pathogenic human OPA1 in their GABAergic motor neurons: axonal mitochondria number, neuronal cell death and defecation cycle time. The described methods can deepen our understanding of ADOA pathogenesis and offer a practical framework for developing novel treatment schemes, providing hope for improved therapeutic outcomes and a better quality of life for individuals affected by this currently incurable condition.

Meeting Summary of The NYO3 5th NO-Age/AD Meeting and the 1st Norway-UK Joint Meeting on Aging and Dementia: Recent Progress on the Mechanisms and Interventional Strategies.

Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a "Money Follows Cooperation agreement" between the 2 nations. The inaugural Norway-UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging.

Chemotaxis assay for evaluation of memory-like behavior in wild-type and Alzheimer's-disease-like C. elegans models.

Here, we present an olfactory-dependent chemotaxis assay for evaluating changes in memory-like behavior in both wild-type and Alzheimer's-disease-like C. elegans models. We describe steps for synchronizing and preparing C. elegans populations and for performing isoamyl alcohol conditioning during starvation and chemotaxis assaying. We then detail counting and quantification procedures. This protocol is applicable to mechanistic exploration and drug screening in neurodegenerative diseases and brain aging.

Caenorhabditis elegans as a Model System to Study Human Neurodegenerative Disorders.

In recent years, advances in science and technology have improved our quality of life, enabling us to tackle diseases and increase human life expectancy. However, longevity is accompanied by an accretion in the frequency of age-related neurodegenerative diseases, creating a growing burden, with pervasive social impact for human societies. The cost of managing such chronic disorders and the lack of effective treatments highlight the need to decipher their molecular and genetic underpinnings, in order to discover new therapeutic targets. In this effort, the nematode Caenorhabditis elegans serves as a powerful tool to recapitulate several disease-related phenotypes and provides a highly malleable genetic model that allows the implementation of multidisciplinary approaches, in addition to large-scale genetic and pharmacological screens. Its anatomical transparency allows the use of co-expressed fluorescent proteins to track the progress of neurodegeneration. Moreover, the functional conservation of neuronal processes, along with the high homology between nematode and human genomes, render C. elegans extremely suitable for the study of human neurodegenerative disorders. This review describes nematode models used to study neurodegeneration and underscores their contribution in the effort to dissect the molecular basis of human diseases and identify novel gene targets with therapeutic potential.

The Role of Mitophagy in Skeletal Muscle Damage and Regeneration.

Mitochondria are cellular organelles that play an essential role in generating the chemical energy needed for the biochemical reactions in cells. Mitochondrial biogenesis, i.e., de novo mitochondria formation, results in enhanced cellular respiration, metabolic processes, and ATP generation, while autophagic clearance of mitochondria (mitophagy) is required to remove damaged or useless mitochondria. The balance between the opposing processes of mitochondrial biogenesis and mitophagy is highly regulated and crucial for the maintenance of the number and function of mitochondria as well as for the cellular homeostasis and adaptations to metabolic demands and extracellular stimuli. In skeletal muscle, mitochondria are essential for maintaining energy homeostasis, and the mitochondrial network exhibits complex behaviors and undergoes dynamic remodeling in response to various conditions and pathologies characterized by changes in muscle cell structure and metabolism, such as exercise, muscle damage, and myopathies. In particular, the involvement of mitochondrial remodeling in mediating skeletal muscle regeneration following damage has received increased attention, as modifications in mitophagy-related signals arise from exercise, while variations in mitochondrial restructuring pathways can lead to partial regeneration and impaired muscle function. Muscle regeneration (through myogenesis) following exercise-induced damage is characterized by a highly regulated, rapid turnover of poor-functioning mitochondria, permitting the synthesis of better-functioning mitochondria to occur. Nevertheless, essential aspects of mitochondrial remodeling during muscle regeneration remain poorly understood and warrant further characterization. In this review, we focus on the critical role of mitophagy for proper muscle cell regeneration following damage, highlighting the molecular mechanisms of the mitophagy-associated mitochondrial dynamics and network reformation.

The Crosstalk between Microbiome and Mitochondrial Homeostasis in Neurodegeneration.

Mitochondria are highly dynamic organelles that serve as the primary cellular energy-generating system. Apart from ATP production, they are essential for many biological processes, including calcium homeostasis, lipid biogenesis, ROS regulation and programmed cell death, which collectively render them invaluable for neuronal integrity and function. Emerging evidence indicates that mitochondrial dysfunction and altered mitochondrial dynamics are crucial hallmarks of a wide variety of neurodevelopmental and neurodegenerative conditions. At the same time, the gut microbiome has been implicated in the pathogenesis of several neurodegenerative disorders due to the bidirectional communication between the gut and the central nervous system, known as the gut-brain axis. Here we summarize new insights into the complex interplay between mitochondria, gut microbiota and neurodegeneration, and we refer to animal models that could elucidate the underlying mechanisms, as well as novel interventions to tackle age-related neurodegenerative conditions, based on this intricate network.

Age-dependent nuclear lipid droplet deposition is a cellular hallmark of aging in Caenorhabditis elegans.

Aging is the major risk factor for several life-threatening pathologies and impairs the function of multiple cellular compartments and organelles. Age-dependent deterioration of nuclear morphology is a common feature in evolutionarily divergent organisms. Lipid droplets have been shown to localize in most nuclear compartments, where they impinge on genome architecture and integrity. However, the significance of progressive nuclear lipid accumulation and its impact on organismal homeostasis remain obscure. Here, we implement non-linear imaging modalities to monitor and quantify age-dependent nuclear lipid deposition in Caenorhabditis elegans. We find that lipid droplets increasingly accumulate in the nuclear envelope, during aging. Longevity-promoting interventions, such as low insulin signaling and caloric restriction, abolish the rate of nuclear lipid accrual and decrease the size of lipid droplets. Suppression of lipotoxic lipid accumulation in hypodermal and intestinal nuclei is dependent on the transcription factor HLH-30/TFEB and the triglyceride lipase ATGL-1. HLH-30 regulates the expression of ATGL-1 to reduce nuclear lipid droplet abundance in response to lifespan-extending conditions. Notably, ATGL-1 localizes to the nuclear envelope and moderates lipid content in long-lived mutant nematodes during aging. Our findings indicate that the reduced ATGL-1 activity leads to excessive nuclear lipid accumulation, perturbing nuclear homeostasis and undermining organismal physiology, during aging.

Mitophagy and Neuroinflammation: A Compelling Interplay.

Mitochondria are the main sites of energy production and a major source of metabolic stress. Not surprisingly, impairment of mitochondrial homeostasis is strongly associated with the development and progression of a broad spectrum of human pathologies, including neurodegenerative disorders. Mitophagy mediates the selective degradation of damaged organelles, thus promoting cellular viability and tissue integrity. Defective mitophagy triggers cellular senescence and prolonged neuroinflammation, leading eventually to cell death and brain homeostasis collapse. Here, we survey the intricate interplay between mitophagy and neuroinflammation, highlighting that mitophagy can be a focal point for therapeutic interventions to tackle neurodegeneration.

The compartmentalised nature of neuronal mitophagy: molecular insights and implications.

The maintenance of a healthy mitochondrial network and the ability to adjust organelle population in response to internal or external stimuli are essential for the function and the survival of eukaryotic cells. Over the last two decades several studies have demonstrated the paramount importance of mitophagy, a selective form of autophagy that removes damaged and/or superfluous organelles, in organismal physiology. Post-mitotic neuronal cells are particularly vulnerable to mitochondrial damage, and mitophagy impairment has emerged as a causative factor in multiple neurodegenerative pathologies, including Alzheimer's disease and Parkinson's disease among others. Although mitochondrial turnover is a multifaceted process, neurons have to tackle additional complications, arising from their pronounced bioenergetic demands and their unique architecture and cellular polarisation that render the degradation of distal organelles challenging. Mounting evidence indicates that despite the functional conservation of mitophagy pathways, the unique features of neuronal physiology have led to the adaptation of compartmentalised solutions, which serve to ensure seamless mitochondrial removal in every part of the cell. In this review, we summarise the current knowledge concerning the molecular mechanisms that mediate mitophagy compartmentalisation and discuss their implications in various human pathologies.

Assessment of dopaminergic neuron degeneration in a C. elegans model of Parkinson's disease.

Transgenic Caenorhabditis elegans that expresses the full-length wild-type human α-synuclein in dopaminergic neurons provides a well-established Parkinson's disease (PD) nematode model. Here, we present a detailed protocol to monitor and dissect the molecular underpinnings of age-associated neurodegeneration using this PD nematode model. This protocol includes preparation of nematode growth media and bacterial food sources, as well as procedures for nematode growth, synchronization, and treatment. We then describe procedures to assess dopaminergic neuronal death in vivo using fluorescence imaging. For complete details on the use and execution of this protocol, please refer to SenGupta et al. (2021).

Amelioration of Alzheimer's disease pathology by mitophagy inducers identified via machine learning and a cross-species workflow.

A reduced removal of dysfunctional mitochondria is common to aging and age-related neurodegenerative pathologies such as Alzheimer's disease (AD). Strategies for treating such impaired mitophagy would benefit from the identification of mitophagy modulators. Here we report the combined use of unsupervised machine learning (involving vector representations of molecular structures, pharmacophore fingerprinting and conformer fingerprinting) and a cross-species approach for the screening and experimental validation of new mitophagy-inducing compounds. From a library of naturally occurring compounds, the workflow allowed us to identify 18 small molecules, and among them two potent mitophagy inducers (Kaempferol and Rhapontigenin). In nematode and rodent models of AD, we show that both mitophagy inducers increased the survival and functionality of glutamatergic and cholinergic neurons, abrogated amyloid-ß and tau pathologies, and improved the animals' memory. Our findings suggest the existence of a conserved mechanism of memory loss across the AD models, this mechanism being mediated by defective mitophagy. The computational-experimental screening and validation workflow might help uncover potent mitophagy modulators that stimulate neuronal health and brain homeostasis.

Sustained intracellular calcium rise mediates neuronal mitophagy in models of autosomal dominant optic atrophy.

Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca2+-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca2+ levels, and decreased axonal mitochondrial density. Chelation of Ca2+ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca2+ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca2+-calcineurin-AMPK signaling cascade.

Autophagy in healthy aging and disease.

Autophagy is a fundamental cellular process that eliminates molecules and subcellular elements, including nucleic acids, proteins, lipids and organelles, via lysosome-mediated degradation to promote homeostasis, differentiation, development and survival. While autophagy is intimately linked to health, the intricate relationship among autophagy, aging and disease remains unclear. This Review examines several emerging features of autophagy and postulates how they may be linked to aging as well as to the development and progression of disease. In addition, we discuss current preclinical evidence arguing for the use of autophagy modulators as suppressors of age-related pathologies such as neurodegenerative diseases. Finally, we highlight key questions and propose novel research avenues that will likely reveal new links between autophagy and the hallmarks of aging. Understanding the precise interplay between autophagy and the risk of age-related pathologies across organisms will eventually facilitate the development of clinical applications that promote long-term health.

Alteration of mitochondrial homeostasis is an early event in a C. elegans model of human tauopathy.

Tauopathies are a group of progressive neurodegenerative disorders characterized by the presence of insoluble intracellular tau filaments in the brain. Evidence suggests that there is a tight connection between mitochondrial dysfunction and tauopathies, including Alzheimer's disease. However, whether mitochondrial dysfunction occurs prior to the detection of tau aggregates in tauopathies remains elusive. Here, we utilized transgenic nematodes expressing the full length of wild type tau in neuronal cells and monitored mitochondrial morphology alterations over time. Although tau-expressing nematodes did not accumulate detectable levels of tau aggregates during larval stages, they displayed increased mitochondrial damage and locomotion defects compared to the control worms. Chelating calcium restored mitochondrial activity and improved motility in the tau-expressing larvae suggesting a link between mitochondrial damage, calcium homeostasis and neuronal impairment in these animals. Our findings suggest that defective mitochondrial function is an early pathogenic event of tauopathies, taking place before tau aggregation and undermining neuronal homeostasis and organismal fitness. Understanding the molecular mechanisms causing mitochondrial dysfunction early in tauopathy will be of significant clinical and therapeutic value and merits further investigation.

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

Aging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., α-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.