Christopher Rongo et al. (1999) International C. elegans Meeting "UNC-43 CaMKII regulates the density of central glutamatergic synapses in vivo"

Joshua Kaplan, Christopher Rongo

[International C. elegans Meeting, 1999]

Synaptic connections undergo a dynamic process of stabilization or elimination during development. To determine how central synapses change during development, we observed neuron-neuron synapses in C. elegans that contain the AMPA-type GluR GLR-1. We previously showed that chimeric receptors tagged with the GFP (GLR-1::GFP) can be used to visualize glutamatergic synapses. In late L1 animals, GLR-1::GFP becomes localized to synapses along the lengths of axons in both the ventral cord and nerve ring processes. During larval growth, the density of GLR-1 synapses is held constant despite dramatic increases in neurite length. Here we describe the role of the UNC-43 type II calcium and calmodulin dependent protein kinase (CaMKII; D. Reiner, E. Newton, & J. Thomas, pers. comm.) in regulating formation of glutamatergic synapses in C. elegans. The coupling of synapse number to neurite length requires both UNC-43, and the UNC-2 and EGL-19 voltage-gated calcium channel subunits. Mutants lacking UNC-43, UNC-2, or EGL-19 accumulate high levels of perinuclear GLR-1::GFP in their cell bodies. Constitutive activation of UNC-43 also resulted in reduced numbers of GLR-1-synapses; however, GLR-1::GFP did not accumulate in the cell bodies of neurons. A GFP::UNC-43 protein containing a mutation that generates a constitutively active kinase was diffusely distributed in axons, unlike wild-type GFP::UNC-43, which was localized to punctate structures in axons. Thus, constitutively activated CaMKII fails to localize to synaptic sites and decreases the density of GLR-1 synapses, suggesting that synaptically localized CaMKII is required to add or maintain GLR-1 at synaptic sites. Our results suggest that CaMKII regulates GLR-1 by two distinct mechanisms: regulating transport of GLR-1 from cell bodies to axons, and regulating the addition or maintenance of GLR-1 to synapses.

Liu, Gang et al. (2009) International Worm Meeting "THE EGF/RAS/MAPK PATHWAY AND THE UFD COMPLEX UPREGULATE THE UBIQUITIN-PROTEASOME SYSTEM AS ADULT NEMATODES MATURE Gang Liu and Christopher Rongo, The Waksman Institute, Department of Genetics, Rutgers University, Piscataway, NJ 08854."

Liu, Gang, Rongo, Christopher

[International Worm Meeting, 2009]

The Ubiquitin-Proteasome System (UPS) is an important regulatory mechanism used to control acute protein turnover and scavenge misfolded proteins in numerous cellular processes. Indeed, the failure of the UPS to remove misfolded proteins is believed to contribute to aging as well as several neurodegenerative disorders. Although recent studies have explored the biochemical function and protein-protein interactions of the UPS, regulated changes in UPS activity have been difficult to observe in vivo in specific tissues as animals develop and age. Here we have employed the GFP reporter UbG76V-GFP, which was developed for monitoring UPS activity, to study the tissue- and age-specific regulation of proteolysis in C. elegans. The UbG76V-GFP reporter contains a mutated ubiquitin fused to GFP, and is a substrate for polyubiquitination and degradation by the 26S proteasome (1). Using tissue specific promoters, we have expressed UbG76V-GFP in C. elegans, where it is a substrate for the UPS. Mutations in the ubiquitin moiety that prevent K48 and K63 polyubiquitin chain addition block turnover of the reporter. Mutations and RNAi treatment that reduce 26S Proteasome activity also block reporter turnover, confirming the in vivo functionality of this UPS reporter. Interestingly, the UbG76V-GFP reporter is stable in the hypodermal tissue of larvae, indicating that growing animals maintain low levels of proteasome activity. Upon maturation, adult animals displayed an age-specific decrease in reporter stability (with no change in the levels of an mRFP internal control expressed from the same promoter), indicating an upregulation of UPS activity. The UFD complex, including the E3 ligase CHN-1 and the E4 enzyme UFD-2 (2), are required for this increase in UPS activity in maturing adults. Mutations that reduce UFD gene activity do not appear to grossly affect larval health, but greatly shorten the lifespan of adult nematodes, suggesting that the upregulation of UPS activity is critical for long-term viability. We find that EGF signaling through the Ras-MAPK pathway and the transcription factors EOR-1 and EOR-2 (3) are required for this change in UPS activity. Our results suggest that mature animals use the EGF/Ras/MAPK signaling pathway to upregulate UPS activity in anticipation of an increased burden of misfolded proteins during aging. References Dantuma et al., (2000) Nature Biotechnology 18:538-43. Hoppe et al., (2004) Cell 118:337-49. Howard et al., (2002) Genes & Development 16:1815-27.

Bowerman BA et al. (1994) Worm Breeder's Gazette "The Maternal Gene skn-4 and the Specification of Ventral Blastomere Fates in the Early C. elegans Embryo"

Bowerman BA, Shelton CA, Thorpe CJ, Martin PR

[Worm Breeder's Gazette, 1994]

THE MATERNAL GENE SKN-4 AND THE SPECIFICATION OF VENTRAL BLASTOMERE FATES IN THE EARLY C. ELEGANS EMBRYO Bruce Bowerman, Paula R. Martin, Christopher J. Thorpe, and Christopher A. Shelton. The Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

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.

Zhang, Donglei et al. (2009) International Worm Meeting "THE ROLE OF RAB SMALL GTPASES IN GLUTAMATE RECEPTOR TRAFFICKING."

Rongo, Christopher, Zhang, Donglei, Isack, Nora, Glodowski, Doreen

[International Worm Meeting, 2009]

Regulated endocytosis and trafficking of AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combinations of clathrin-dependent and -independent mechanisms that mediate AMPAR trafficking in vivo have not been fully characterized. To better understand AMPAR trafficking, we have been examining the trafficking of the AMPAR GLR-1 in C. elegans (1). GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit (2). We previously identified two genes that regulate GLR-1 membrane recycling: RAB-10 and LIN-10 (2, 3). Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, or LIN-10, a PDZ-domain containing protein, share the same phenotype: GLR-1 accumulates in large, internalized accretions and animals display a decreased frequency of reversals (3, 4). Interestingly, reducing clathrin-dependent endocytosis specifically suppresses the lin-10 mutant phenotype, whereas reducing clathrin-independent endocytosis specifically suppresses the rab-10 mutant phenotype. Thus, we hypothesize that LIN-10 and RAB-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways. Moreover, we suspect that another Rab protein, analogous to RAB-10, functions in the LIN-10 pathway. We have taken two approaches to identify additional cellular factors involved in LIN-10-mediated trafficking of GLR-1. First, we have performed a yeast 2-hybrid screen using LIN-10 as bait. We are characterizing the functions of the proteins identified in the screen to determine if they regulate AMPAR trafficking. Second, we are testing all of the known Rab genes in the genome for a role in GLR-1 trafficking, either by analyzing previously identified mutations or by generating mutant transgenes that express GDP- or GTP-locked versions of the Rabs. Our long-term goal is to further define the regulatory pathways involved in the movement and localization of AMPARs at synapses in vivo. 1. Hart, A.C. et al., Nature 378, 82-85 (1995) and Maricq, A.V. et al., Nature 378, 78-81 (1995). 2. Rongo, C. et al., Cell 94, 751-759 (1998). 3. Glodowski, D. et al., Mol Biol Cell 18, 4387-96 (2007). 4. Chen et al., Mol Biol Cell 17, 1286-97 (2006).

Joshi, Kishore et al. (2015) International Worm Meeting "Dopamine Signaling Regulates Protein Homeostasis Through The Ubiquitin Proteasome Systems."

Rongo, Christopher, Matlack, Tarmie, Joshi, Kishore

[International Worm Meeting, 2015]

Protein homeostasis is a key facet of longevity, and failure in protein quality control is linked to both age-associated decline and neurodegenerative disorders. One key regulator of protein homeostasis is the Ubiquitin-Proteasome System (UPS), which acts globally to degrade damaged and oxidized proteins. Multiple hormonal and growth factor signaling pathways are implicated in regulating longevity, but the precise interaction between these signaling pathways, global UPS activity, protein homeostasis, and aging is not well understood. We recently demonstrated that components of the Ubiquitin Fusion Degradation (UFD) pathway promote longevity in C. elegans by mediating the polyubiquitination and degradation of damaged and unfolded proteins. We also showed that growth factor signaling modulates the level of global UPS activity at different points during adult maturation and aging, thereby promoting longevity and health. Using an in vivo reporter for UPS activity, we have now performed an RNAi screen (followed by validation with true genetic mutants) for additional regulators of global UPS and identified a dopamine (DA) receptor as a regulator of UPS activity in maturing adults. Here we analyze the detailed molecular interactions between DA signaling and UPS activation in maturing adults by targeting genes involved in DA synthesis, release, reception, and signal transduction. We find that DA is used as an extracellular neurohormone to adjust UPS activity in specific tissues and at specific points in development and aging. Moreover, our recent data shows that specific set of dopamine receptors attribute to this UPS activity regulation in distal tissues and contributes in organismal aging. Molecules involved in this DA-mediated regulation of the UPS are thus potential therapeutic targets for minimizing age-associated decline and neurological disorders.

Joshi, Kishore K et al. (2013) International Worm Meeting "Modulation of the Ubiquitin-Proteasome System and C. elegans Longevity by Neuroendocrine and Growth Factor Signaling Pathways."

Rongo, Christopher, Shah, Tejash, Joshi, Kishore K

[International Worm Meeting, 2013]

Protein homeostasis is a key facet of longevity, and failure in protein quality control is linked to both age-associated decline and neurodegenerative disorders. One key regulator of protein homeostasis is the Ubiquitin-Proteasome System (UPS), which acts globally to degrade damaged and oxidized proteins. Multiple hormonal and growth factor signaling pathways are implicated in regulating longevity, but the precise interaction between these signaling pathways, global UPS activity, protein homeostasis, and aging is not well understood. We recently demonstrated that components of the Ubiquitin Fusion Degradation (UFD) pathway promote longevity in C. elegans by mediating the polyubiquitination and degradation of damaged and unfolded proteins. We also showed that growth factor signaling modulates the level of global UPS activity at different points during adult maturation and aging, thereby promoting longevity and healthspan. Using an in vivo reporter for UPS activity, we have now performed an RNAi screen (followed by validation with true genetic mutants) for additional regulators of global UPS and identified a dopamine (DA) receptor as a regulator of UPS activity in maturing adults. Here we analyze the detailed molecular interactions between DA signaling and UPS activation in maturing adults by targeting genes involved in DA synthesis, release, reception, and signal transduction. We find that DA is used as an extracellular neurohormone to adjust UPS activity in specific tissues and at specific points in development and aging. Molecules involved in this DA-mediated regulation of the UPS are thus potential therapeutic targets for minimizing age-associated decline and neurological disorders.

Liu, Gang et al. (2011) International Worm Meeting "Epidermal Growth Factor Signaling Activates The Ubiquitin Proteasome System To Modulate C. elegans Lifespan."

Rongo, Christopher, Liu, Gang, Murphy, Coleen, Rogers, Jason

[International Worm Meeting, 2011]

Epidermal growth factor (EGF) signaling regulates cell growth, proliferation, and differentiation, and has recently been implicated in regulating lifespan, although the mechanism remains unclear. Here we examine the function of EGF signaling in lifespan in adult C. elegans. We find that EGF signaling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. We find that as animals enter adulthood, EGF signaling upregulates the expression of genes involved in the ubiquitin/proteasome system (UPS), including the Skp1-like protein SKR-5 (an adaptor between F-box proteins and Cullin scaffolding in SCF ubiquitin ligases), while downregulating the expression of HSP16-type chaperones. Using GFP-based reporters for (1) global UPS activity, (2) protein aggregation, and (3) oxidative stress, we find that EGF signaling alters protein homeostasis in adults, triggering an increase in UPS activity and in the levels of polyubiquitinated proteins, while also increasing the propensity of misfolded proteins to aggregate - critical facets in aging and cellular degeneration. We show that SKR-5 and the components of the E3/E4 ligases that comprise the Ubiquitin Fusion Degradation (UFD) complex are required for the increase in UPS activity observed in adults, suggesting a novel mechanism - regulation of Skp1 adaptor protein expression - by which cells can regulate the activity of multiple E3 ubiquitin ligases. Animals that fail to upregulate UPS activity via EGF signaling, SKR-5, or the UFD have reduced lifespans and indications of oxidative stress, whereas a mutation that overactivates the EGF receptor prematurely accelerates UPS activity and increases lifespan. Taken together, our results indicate that as animals enter adulthood, EGF signaling switches the mechanism for maintaining protein homeostasis from a chaperone-based approach to an approach involving protein elimination via augmented UPS activity.

Zhang, Donglei et al. (2011) International Worm Meeting "RAB-6 and the Retromer Complex Regulate Glutamate Receptor Recycling Through A Retrograde Transport Pathway."

Zhang, Donglei, Isack, Nora R., Rongo, Christopher, Grant, Barth D., Glodowski, Doreen R.

[International Worm Meeting, 2011]

Synaptic plasticity is regulated by the collective endocytosis, membrane recycling, and degradation of AMPA-type glutamate receptors (AMPARs), yet the specific intracellular trafficking pathways available to AMPARs, and the mechanism by which such pathways are selected for AMPARs in neurons, is poorly understood. Here we show that the AMPAR subunit GLR-1 in C. elegans utilizes the retrograde transport pathway to regulate its synaptic abundance. GLR-1 is localized on post-synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit. GLR-1 synaptic abundance is regulated by a combination of endocytosis and membrane recycling, although the specific pathways have not been fully elucidated. One possible pathway is retrograde transport via Rab6 GTPases and the retromer complex, which shepherd certain membrane proteins from endosomes back to Golgi; in the absence of retrograde transport, cargo proteins are degraded. We find that in mutants for rab-6.2, or the retromer genes vps-35, snx-1, and rme-8, GLR-1::GFP receptors are degraded in neurons, and mutant animals show behavioral defects indicative of diminished GLR-1 function. By contrast, expression of a constitutively active RAB-6.2 drives the retrograde transport of GLR-1 back to cell body Golgi. Mutations that block endocytosis suppress the effects of rab-6.2 mutations, suggesting that RAB-6.2 functions downstream of GLR-1 endocytosis. Mutations that block GLR-1 turnover in rab-6.2 mutants cause the accumulation of GLR-1 in endosomes, suggesting that GLR-1 is directed to lysosomes when retrograde trafficking is blocked. MIG-14, which undergoes retrograde recycling, undergoes a similar degradation in rab-6.2 mutants, consistent with a loss of retrograde transport. In its GTP-bound state, RAB-6.2 physically interacts with, colocalizes with, and directs the subcellular localization of the PDZ/PTB domain protein LIN-10, and the regulation of GLR-1 retrograde transport by RAB-6.2 requires LIN-10 activity. Specific retrograde cargo and a function for the retromer in neurons have not been previously demonstrated. Our results indicate that RAB-6.2 and LIN-10 recycle AMPARs along a retrograde transport pathway in neurons so as to maintain synaptic strength.

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.