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.
Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.
Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.
Phagocytosis of dying cells is critical in development and immunity1–3. While proteins for recognition and engulfment of cellular debris following cell death are known4,5, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically-complex cells, such as neurons, glia, and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments6,7. Such cells, therefore, offer unique settings to explore engulfment program mechanisms and specificity. Here we report that dismantling and clearance of a morphologically-complex C. elegans epithelial cell requires separate cell-soma, proximal-, and distal-process programs. Similar compartment-specific events govern elimination of a C. elegans neuron. While canonical engulfment proteins drive cell-soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell-cell fusion8, specifically promotes distal-process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips, and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically-complex cells, and uncover a phagosome sealing component promoting cell-process clearance.
Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies have delineated a bewildering number of cell death mechanisms, most have only been observed in pathological contexts, and only a small number drive normal development. This Primer outlines the important roles, different types and molecular players regulating developmental cell death, and discusses recent findings with which the field currently grapples. We also clarify terminology, to distinguish between developmental cell death mechanisms, for which there is evidence for evolutionary selection, and cell death that follows genetic, chemical or physical injury. Finally, we suggest how advances in understanding developmental cell death may provide insights into the molecular basis of developmental abnormalities and pathological cell death in disease.
Programmed cell death (PCD) is a common cell fate in metazoan development. PCD effectors are extensively studied, but how they are temporally regulated is less understood. Here we report a mechanism controlling tail-spike cell death onset during C. elegans development. We show that the Zn-finger transcription factor BLMP-1/Blimp1, which controls larval development timing, also regulates embryonic tail-spike cell death initiation. BLMP-1 functions upstream of CED-9/BCL-2 and in parallel to DRE-1/FBXO11, another CED-9 and tail-spike cell death regulator. BLMP-1 expression is detected in the tail-spike cell shortly after the cell is born, and blmp-1 mutations promote ced-9-dependent tail-spike cell survival. BLMP-1 binds ced-9/bcl-2 gene regulatory sequences, and inhibits ced-9 transcription just before cell-death onset. BLMP-1 and DRE-1 function together to regulate developmental timing, and their mammalian homologs regulate B-lymphocyte fate. Our results, therefore, identify roles for developmental timing genes in cell-death initiation, and suggest conservation of these functions.
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