Ghose, Piya et al. (2009) International Worm Meeting "The Effect of Hypoxia on Neuronal Necrosis and Glutamate Receptor Trafficking."

Rongo, Christopher, Ghose, Piya, Park, Eun Chan

[International Worm Meeting, 2009]

Neurons in the brain are sensitive to oxygen levels, undergoing necrosis within minutes after hypoxia (e.g., oxygen deprivation during ischemic stroke). This neuronal toxicity is mediated in part by the excitatory neurotransmitter glutamate, which is released at high levels during hypoxia, and the ionotropic glutamate receptors (GluRs) that are trafficked to postsynaptic membranes. However, the mechanistic relationship between hypoxia, GluR function and subcellular trafficking, and excitotoxic necrosis has not been fully elucidated. Here we examine the effects of hypoxia and mutations in hypoxia response factors on necrosis and GluR trafficking in C. elegans. In C. elegans, an excitotoxic-like necrosis can be modeled in vivo by expressing a constitutively active G alpha S subunit in neurons. Activated G alpha S modulates the activity of yet unknown ion channels, resulting in the necrosis of a subset of neurons that express it (1). The resulting intermediate necrosis phenotype affords us the ability to screen for factors that either enhance or suppress neuronal necrosis, including hypoxia. The C. elegans GluR GLR-1 is expressed in these same neurons, where it functions to mediate locomotion reversal behavior (2,3) and is trafficked to synaptic connections (4). We find that hypoxic treatment enhances the necrosis caused by activated G alpha S expression, and alters the trafficking of GLR-1 in these same neurons. We anticipate that our studies will further elucidate the mechanisms of neuronal damage after hypoxia. By identifying the molecular mechanisms that regulate GluR signaling and necrosis in response to hypoxia, we hope to provide useful therapeutic targets for the prevention of nervous system damage after hypoxia exposure.(*co-first author) References 1. Berger, A.J. et al., G alphas-induced neurodegeneration in Caenorhabditis elegans. J Neurosci. 18,2871-80 (1995). 2. Hart, A.C. et al., Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature. 378,82-5 (1995). 3. Maricq, A.V. et al., Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature. 378,78-81 (1995). 4. Rongo, C. et al., LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell 94,51-759, 1998.

Ghose, Piya et al. (2017) International Worm Meeting "The fusogen EFF-1 promotes a novel cell process-specific engulfment program."

Singhal, Anupriya, Insley, Peter, Trivedi, Meera, Ghose, Piya, Shah, Pavak, Bao, Zhirong, Lu, Yun, Shaham, Shai

[International Worm Meeting, 2017]

Programmed cell death and cell process pruning are common in metazoan development and homeostasis. Dismantling of morphologically complex cells, sporting long processes, poses a particularly interesting problem, as different regions of a cell are often in different microenvironments, and contact different cells. We have used the C. elegans tail-spike cell (TSC) to understand the death and clearance of a morphologically complex cell. The TSC extends a microtubule-laden process towards the tail tip during embryonic morphogenesis and then dies. TSC ablation reveals that this cell promotes tail morphogenesis. Tracking of TSC demise using still images and long-term light-sheet microscopy reveals that this morphologically complex cell undergoes three distinct degeneration/clearance events. The proximal TSC process is dismantled first, and undergoes beading reminiscent of Wallerian degeneration. The cell soma then dies and is cleared in a manner resembling other apoptotic cells. The distal process retracts and accumulates in a distinct varicosity. All three mechanisms depend independently on CED-3/caspase. Importantly, a similar sequence accompanies the demise and clearance of embryonic CEM neurons in hermaphrodites. Thus, the TSC death program may represent a general mechanism for dismantling morphologically complex cells, including neurons. Clearance of the proximal and distal TSC fragment is independent of known apoptotic clearance proteins, except for SAND-1, which promotes phagosome maturation, suggesting that novel mechanisms are at play. From a genetic screen for mutants disrupting TSC clearance, we identified two mutants with lesions in eff-1, encoding a previously-described C. elegans fusogen. Mutant animals, and animals carrying the canonical eff-1(hy21) allele, are defective in TSC distal process varicosity clearance, and do not display cell body or proximal process clearance defects. EFF-1 is expressed in and functions in hyp10 cells, which surround and engulf TSC process remnants. While hyp10 appears to recognize the distal process varicosity, the phagosome that is formed seems not to be closed, suggesting that EFF-1 may mediate the fusion event required for membrane scission and phagosome sealing. Supporting this idea, the undegraded open phagosome is labeled with PLCd-PH::mKate2, a reporter for unsealed phagosomes. Furthermore, a fusion-dead version of EFF-1 fails to rescue distal process clearance. EFF-1 also localizes to puncta adjacent to the TSC remnant, perhaps corresponding to the site of coalescing phagosome arms. Direct mediators of membrane scission that promote the formation of endosomes, phagosomes, and other plasma membrane-derived organelles are not known. Our data reveal a novel paradigm for complex cell dismantling that may be broadly conserved, and suggest a novel role for EFF-1 in the scission event promoting phagosome sealing.

Ghose, Piya et al. (2015) International Worm Meeting "Compartment-specific killing and clearance programs in the C. elegans tail-spike cell."

Lu, Yun, Trivedi, Meera, Ghose, Piya, Shaham, Shai, Insley, Peter

[International Worm Meeting, 2015]

While distinct programs are hypothesized to mediate degeneration of different parts of a cell, the precise cell biological and molecular events leading to compartment-specific destruction, such as neurite degeneration and pruning, are not well understood. We address this in the C. elegans tail-spike cell, a structurally complex cell that dies during embryonic development. This cell resembles a neuron, projecting an axon-like process towards the tail tip. Electron and fluorescence microscopy reveal that the tail-spike cell can be divided into three compartments: the cell body, the process proximal to the cell body, and a distal process segment. Scanning light-sheet microscopy indicates that the three compartments undergo region-specific degeneration with features resembling apoptotic death and clearance, axon fragmentation similar to that observed in Wallerian degeneration, and cytoplasmic transport and removal, respectively. We previously showed that cell body and process degeneration require CED-3/caspase and its adapter CED-4/Apaf-1, but are largely independent of EGL-1/BH3-only. Here we describe tail-spike cell compartment-specific killing and clearance programs. Importantly, we show that animals carrying a weak ced-3/caspase mutation can exhibit intact tail-spike cell soma or process alone, suggesting independent control of cell body and process degeneration by caspases. We also find that while ced-5/DOCK180 controls only cell body and not process engulfment, the conserved phagosome maturation gene sand-1 controls degradation of both, suggesting that independent engulfment programs converge on a common degradation machinery. We further report that the cell fusion receptor EFF-1, known to cell-autonomously mediate axon regeneration and dendrite sculpting, acts in surrounding cells to specifically promote degradation of the tail-spike cell distal process. Our studies uncover important roles for conserved genes in compartment-selective killing and degradation that are consistent with reports of differential requirements for process and cell body death and engulfment in other animals. We thus identify the tail-spike cell as an exciting and novel venue for understanding the cellular and molecular events governing complex cell elimination.

Tabakin, Alexandra L. et al. (2013) International Worm Meeting "Mitochondrial dynamics in response to oxygen deprivation."

Salazar-Vasquez, Nathaly, Ghose, Piya, Park, Eun Chan, Rongo, Christopher, Tabakin, Alexandra L.

[International Worm Meeting, 2013]

Mitochondria play a critical role in the oxygen-dependent generation of cellular energy. Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. How mitochondria are regulated in the face of oxygen deprivation stress is not well understood. Here we examine mitochondrial dynamics, including organelle fission and fusion, in C. elegans neurons in response to anoxia. We find that anoxia triggers mitochondrial fission, and that DRP-1, a key component of the mitochondrial fission machinery, is required for this fission. Subsequent reoxygenation results in mitochondrial refusion. We observed that mutations in egl-9, a prolyl hydroxylase oxygen sensor, resulted in mitochondrial hyperfusion (fusion beyond the normal levels present prior to anoxia) upon reoxygenation. This hyperfusion phenotype requires the hypoxia inducible factor-1 (HIF-1), the canonical substrate of EGL-9. Hyperfusion also requires STL-1, a poorly characterized mitochondrial scaffolding protein. Finally, we found that anoxia triggers a behavioral arrest (suspended animation), and that normal neurological behavior was restored upon reoxygenation. Mutants for egl-9 demonstrated a rapid recovery from suspended animation compared to wild type; this rapid recovery also required HIF-1 and STL-1. Our results suggest that C. elegans neurons modulate mitochondrial fission and fusion in response to stress to help organisms adapt to low oxygen environments and to modify neurological function; moreover, this modulation is regulated and rapidly reversible.

Park, Eun Chan et al. (2013) International Worm Meeting "Mitochondrial Dynamics And Behavioral Plasticity In Response To Oxygen Deprivation Are Linked Through HIF-1."

Salazar-Vasquez, Nathaly, Ghose, Piya, Park, Eun Chan, Tabakin, Alexandra, Rongo, Christopher

[International Worm Meeting, 2013]

C. elegans can survive extreme oxygen deprivation (anoxia, < 0.1% O2) by entering into a suspended animation state during which locomotory behavior and development arrest. Upon re-exposure to normoxic conditions (21 % O2), animals resume their behavior and normal development. The underlying mechanism of this stress response is not well understood. Here we show that neuronal mitochondria undergo fission in response to anoxia co-temporaneously with the onset of suspended animation, followed by re-fusion upon re-oxygenation, suggesting that mitochondrial dynamics might contribute to anoxia-induced suspended animation. We find that the hypoxia response pathway, including EGL-9 and HIF-1, regulates the reconstitution of mitochondria following re-oxygenation. Under conditions of normal oxygen, EGL-9 represses the activity of HIF-1, a transcription factor that promotes the expression of hypoxia-response genes. Loss of function mutations in egl-9 result in rapid re-fusion of mitochondria and behavioral recovery from suspended animation following re-oxygenation; both phenotypes require HIF-1 activity. In addition, the mitochondria are significantly larger in egl-9 mutants after re-oxygenation than they were prior to anoxia exposure - a phenotype that resembles the stress-induced mitochondria hyperfusion (SIMH) that has been observed in mammalian cells. Both the changes in mitochondrial dynamics and suspended animation recovery of egl-9 mutants can be rescued by expressing wild-type EGL-9 solely in neurons. Mitochondrial hyperfusion and rapid recovery observed in egl-9 mutants following anoxia are modulated by STL-1, a mammalian stomatin-like protein (SLP2) homologue that is reported to function in SIMH. Our results suggest the existence of a conserved stress response involving changes in mitochondrial fission and fusion, and demonstrate that the behaviors executed by a simple neural circuit can be regulated through changes in mitochondrial dynamics.

Park, Eun Chan et al. (2011) International Worm Meeting "HIF-1 Independent Glutamate Receptor Trafficking as a Novel Neuroprotective Response to Hypoxic Insult."

Shao, Zhiyong, Xu, Shawn, Rongo, Christopher, Kang, Lijun, Park, Eun Chan, Powell-Coffman, Jo Anne, Ghose, Piya

[International Worm Meeting, 2011]

Ischemic stroke results in excitotoxicity via hypoxia, glutamate receptor (GluR) overactivation and deregulated calcium homeostasis. Changes in GluR trafficking can occur in response to hypoxia, but the nature of those changes, whether they protect or exacerbate excitotoxicity, remains controversial. Here we examine how hypoxia and the known hypoxia response pathway alter the trafficking of the C. elegans GluR GLR-1. GLR-1 acts in the command interneurons mediating an escape response by triggering reversals in locomotion. Synaptically localized GLR-1 receptors can be observed using a GLR-1::GFP chimeric protein. Recently, we observed a change in GLR-1 trafficking in response to hypoxic treatment: receptors become trapped in internal endosome-like accretions and animals behave similar to glr-1 knockouts. Loss of function mutations in egl-9, a prolyl hydroxylase that normally inhibits the cellular response to hypoxia by hydroxylating key proline residues on the transcription factor HIF-1, mimics the effects of hypoxia on GLR-1 trafficking and depresses both glutamate-activated currents and GLR-1- mediated behaviors. HIF-1, the canonical substrate of EGL-9 activity, is not required for this effect; instead, we find that EGL-9 physically interacts with LIN-10, a PTB/PDZ-domain protein that promotes GLR-1 membrane recycling. We find that normal oxygen levels induce a specific EGL-9 isoform to colocalize with LIN-10 in dendrites, where it prevents the phosphorylation of LIN-10 by the proline-directed kinase CDK-5. Un-phosphorylated LIN-10 is recruited to endosomes, where it mediates GLR-1 recycling. By contrast, under hypoxic conditions, EGL-9 is inhibited by lack of oxygen, allowing CDK-5 to inhibit LIN-10 by directly phosphorylating its proline-rich N-terminus. This results in LIN-10 delocalization and the trapping of GLR-1 in endosomes. Mutations in either cdk-5 or the proline residues of the CDK-5 phosphorylation sites in LIN-10 block the effects of hypoxia and egl-9 mutations, suggesting a novel kinase regulatory mechanism in which EGL-9 regulates CDK-5 kinase activity by hydroxylating its substrate prolines, thus precluding substrate phosphorylation by CDK-5. We propose that neurons use this novel neuroprotective mechanism to respond to hypoxia by inhibiting receptor membrane recycling so as to reduce GluR synaptic abundance.

Ghose, P. et al. (2019) International Worm Meeting "One cell, many deaths: the orchestrated demise of the C. elegans tail-spike cell."

Pena, S., Shaham, S., Ghose, P.

[International Worm Meeting, 2019]

Programmed cell death (PCD) is vital to development and homeostasis. Many of our cells, including melanocytes, glia and neurons are morphologically complex, bearing long processes. How different subcellular compartments communicate with eachother in these cells, and how cell function is coordinated between these compartments is an interesting yet poorly understood problem. This is especially relevant in the context of PCD, where the entire cell needs to die and be removed efficiently to prevent aberrent cell activity and autoimmune consequences. The C. elegans tail-spike cell (TSC) dies during embryonic development in a fascinating way: it segments into three parts, each degenerating differently (Ghose et al.,2018). The soma rounds up like a simple apoptotic cell. The proximal process segment forms beads, much as in Wallerian degneration of axons. The distal process retracts (resembling axon retraction following nutrient deprivation) into a central swelling. Importantly, an identical pattern of degeneration is seen in the CEM neurons, which die sex specifically in the embryo, suggesting that TSC-like death may be a universal mode of eliminating complex cells. How is cell death inititated and coordinated in the TSC? Our findings show that death requires the caspase CED-3, acting independently in the process and soma. Preliminary studies suggest an intriguing role for CED-3 in mitochondrial trafficking. Death also requires the transcriptional factor BLMP-1/BLIMP-1, which appears to coordinate the elimination of TSC compartments. Canonical apoptosis engulfment genes are not required for TSC process removal. A forward genetic screen and subsequent studies reveal that the cell-cell fusogen EFF-1 is required for phagosome sealing around the distal process fragment following retraction (Ghose et al., 2018). EFF-1 functions in the surrounding hyp10 cell and is localized to phagosome arm tips. Importantly, EFF-1 appears to be the first direct regulator identified of phagosome sealing, a poorly understood step of phagocytosis. To identify additional genes promoting TSC death and degradation, we isolated mutants with defects in various aspects of TSC death and clearance following EMS mutagenesis. These include a mutant for the process retraction step and another phagosome sealing mutant, which may represent a regulator of EFF-1. Our mutant collection, together with our previous studies, make the TSC a powerful model to study novel aspects of PCD and removal in the context of morphologically complex cells. References: Ghose, P., Rashid, A., Insley, P., Trivedi, M., Singhal, A., Shah, P., Lu, Y., Bao, Z., Shaham, S. (2018). EFF-1 Fusogen Promotes Phagosome Sealing During Cell Process Clearance in Caenorhabditis elegans. Nat Cell Biol. doi:101038/s41556-018-0068-5.