No abstract

C. elegans is a powerful model for analysis of the conserved mechanisms that modulate healthy aging. In the aging nematode nervous system, neuronal death and/or detectable loss of processes are not readily apparent, but because dendrite restructuring and loss of synaptic integrity are hypothesized to contribute to human brain decline and dysfunction, we combined fluorescence microscopy and electron microscopy (EM) to screen at high resolution for nervous system changes. We report two major components of morphological change in the aging C. elegans nervous system: 1) accumulation of novel outgrowths from specific neurons, and 2) physical decline in synaptic integrity. Novel outgrowth phenotypes, including branching from the main dendrite or new growth from somata, appear at a high frequency in some aging neurons, but not all. Mitochondria are often associated with age-associated branch sites. Lowered insulin signaling confers some maintenance of ALM and PLM neuron structural integrity into old age, and both DAF-16/FOXO and heat shock factor transcription factor HSF-1 exert neuroprotective functions. hsf-1 can act cell autonomously in this capacity. EM evaluation in synapse-rich regions reveals a striking decline in synaptic vesicle numbers and a dimunition of presynaptic density size. Interestingly, old animals that maintain locomotory prowess exhibit less synaptic decline than same-age decrepit animals, suggesting that synaptic integrity correlates with locomotory healthspan. Our data reveal similarities between the aging C. elegans nervous system and mammalian brain, suggesting conserved neuronal responses to age. Dissection of neuronal aging mechanisms in C. elegans may thus influence the development of brain healthspan-extending therapies.

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening.Electronic supplementary materialThe online version of this article (10.1007/s11357-019-00108-9) contains supplementary material, which is available to authorized users.

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach towards aging-intervention screening.

Caenorhabditis elegans neurons have recently been found to throw out cellular debris for remote degradation and/or storage, adding an "extracellular garbage elimination" option to known intracellular protein and organelle degradation pathways. This Q&A describes initial insights into the biology of seemingly selective protein and organelle elimination by challenged neurons, highlighting mysteries of how garbage is distinguished and sorted in the sending neuron, how the garbage-filled "exophers" appear to elicit degradative responses as they transit neighboring tissue, and how non-digestible materials get thrown out of cells again via processes that may be highly relevant to human neurodegenerative disease mechanisms. Don't we already have a good understanding of protein/organelle degradation strategies? Surprisingly, no. A major theme in the maintenance of cell health is that proteins, which make up about 20% of the cell by weight, must be folded properly for both their own functionality and for preventing misfolded or aggregated proteins from gumming up the works in ways that interfere with other cell activities. Considerable characterization of cell strategies for accomplishing protein quality control has defined chaperone functions (protein folding helpers), the ubiquitin proteasome system (which degrades proteins that are tagged as misfolded or otherwise impaired and ready for destruction), and the autophagy system (which degrades cellular entities including proteins and organelles by targeting defective species to the lysosome for import and degradation) as major cell-intrinsic pathways that keep a cell's overall protein content in good shape.

Caenorhabditis elegans ( C. elegans ) lifespan assays constitute a broadly used approach for investigating the fundamental biology of longevity. Traditional C. elegans lifespan assays require labor-intensive microscopic monitoring of individual animals to evaluate life/death over a period of weeks, making large-scale high throughput studies impractical. The lifespan machine developed by Stroustrup et al . (2013) adapted flatbed scanner technologies to contribute a major technical advance in the efficiency of C. elegans survival assays. Introducing a platform in which large portions of a lifespan assay are automated enabled longevity studies of a scope not possible with previous exclusively manual assays and facilitated novel discovery. Still, as initially described, constructing and operating scanner-based lifespan machines requires considerable effort and expertise. Here we report on design modifications that simplify construction, decrease cost, eliminate certain mechanical failures, and decrease assay workload requirements. The modifications we document should make the lifespan machine more accessible to interested laboratories.

In human neurodegenerative diseases, toxic protein aggregates can spread between neurons to promote pathology. In the transparent genetic animal model C. elegans, stressed neurons can concentrate fluorescently tagged protein aggregates and organelles and extrude them in large, nearly soma-sized, membrane-bound vesicles called exophers that enter neighboring cells. C. elegans exophergenesis may occur by mechanisms analogous to those that enable aggregate spreading in the human brain in neurodegenerative disease. Here we report on aggresome-like biology in stressed C. elegans neurons that influences exophergenesis. We show that C. elegans intermediate filament proteins IFD-1 and IFD-2 can assemble into juxtanuclear structures with characteristics similar to mammalian aggresomes and document that these intermediate filaments are required cell autonomously for efficient exopher production. IFD-concentrating structures expand with age or neuronal stress level, can associate with neurotoxic polyglutamine expansion protein HttQ74, and depend upon orthologs of mammalian adapter proteins, dynein motors, and microtubule integrity for collection of aggregates into juxtanuclear compartments. IFD homolog human neurofilament light chain hNFL can substitute for C. elegans IFD-2 proteins in promoting exopher production, indicating conservation of the capacity of intermediate filaments to influence neuronal extrusion. In sum, we identify an unexpected requirement for specific intermediate filaments, counterparts of human biomarkers of neuronal injury and disease, and major components of Parkinson’s disease Lewy bodies, in large vesicle extrusion from stressed neurons.

Toxic protein aggregates can spread among neurons to promote human neurodegenerative disease pathology. We found that in C. elegans touch neurons intermediate filament proteins IFD-1 and IFD-2 associate with aggresome-like organelles and are required cell-autonomously for efficient production of neuronal exophers, giant vesicles that can carry aggregates away from the neuron of origin. The C. elegans aggresome-like organelles we identified are juxtanuclear, HttPolyQ aggregate-enriched, and dependent upon orthologs of mammalian aggresome adaptor proteins, dynein motors, and microtubule integrity for localized aggregate collection. These key hallmarks indicate that conserved mechanisms drive aggresome formation. Furthermore, we found that human neurofilament light chain (NFL) can substitute for C. elegans IFD-2 in promoting exopher extrusion. Taken together, our results suggest a conserved influence of intermediate filament association with aggresomes and neuronal extrusions that eject potentially toxic material. Our findings expand understanding of neuronal proteostasis and suggest implications for neurodegenerative disease progression.