DJ Hoeppner et al. (1999) International C. elegans Meeting "Toward Understanding Cell-Type-Specific Cell Death Regulation"

MO Hengartner, DJ Hoeppner

[International C. elegans Meeting, 1999]

Our goal is to understand how different cell types regulate programmed cell death in C. elegans . Toward this end, we are defining the genes necessary for Hermaphrodite-Specific Neuron (HSN) death. The HSNs are a pair of motor-neurons that are required for egg-laying. The HSNs die by programmed cell death in males, presumably in response to masculine signals from the sex determination pathway. The HSNs also die in hermaphrodites carrying gain-of function alleles of the egl-1 gene. Hermaphrodites lacking HSNs are defective in egg-laying (Egl), but are viable and display no pleiotropic defects. Thus, egl-1(gf) provides a good background to identify mutations in genes that are specifically required for HSN death, as restoration of HSN function must be achieved by blocking its death. In a screen for mutants that revert the egg-laying defect of egl-1(n1084) , we isolated two mutations, op181 IV and op166 X, that are required for HSN death, but not, as far as we can tell, any other cell death. In addition to suppressing HSN death, op181 and op166 have a common set of pleiotropic defects including uncoordinated movement, lethality with a terminal Clr phenotype, egg-laying defects and male mating defects. We speculate that op181 and op166 are mutations in genes required for complete differentiation in the nervous system.

Hoeppner DJ et al. (1995) International C. elegans Meeting "CED(n2431), A NEW CELL DEATH GENE"

Hengartner MO, Hoeppner DJ

[International C. elegans Meeting, 1995]

The genetic pathway leading to programmed cell death during C. elegans development has been defined largely by direct screening for mutations affecting specific cell deaths. Strong loss-of function (lf) mutations in ced-4 or ced-3 and gain-of-function mutations in ced-9 prevent cell death, while lf mutations in ced-9 lead to ectopic cell deaths in both somatic and germ-line tissues; weak alleles result in extra germ-line deaths while strong alleles result in absolute sterility. We exploited the maternal effect lethality conferred by the moderate lf allele ced-9(n1950n2161) to isolate suppressors that would block the cell death pathway and restore viability. As expected, new alleles of ced-3 and ced-4 were isolated. One additional suppressor, n2431, was isolated that appears to be a novel ced gene. Preliminary data suggest that n2431 maps between dpy-17 and unc-36 on chromosome III. n2431 weakly suppresses cell death in the pharynx and germline in ced-9(n1950n2161) worms. n2431 also suppresses the Egl phenotype of the weak ts allele ced-9(n1653), but not the strong allele ced-9(n1950n2077). However, n2431 does not prevent cell death on its own. We are presently investigating genetic interactions between n2431 and other known genes of the death pathway, and are working toward cloning the gene affected by this mutation.

Hoeppner DJ et al. (2001) Nature "Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans."

Schnabel R, Hoeppner DJ, Hengartner MO

[Nature, 2001]

Genetic studies have identified over a dozen genes that function in programmed cell death (apoptosis) in the nematode Caenorhabditis elegans(1-3). Although the ultimate effects on cell survival or engulfment of mutations in each cell death gene have been extensively described, much less is known about how these mutations affect the kinetics of death and engulfment, or the interactions between these two processes. We have used four-dimensional-Nomarski time-lapse video microscopy to follow in detail how cell death genes regulate the extent and kinetics of apoptotic cell death and removal in the early C. elegans embryo. Here we show that blocking engulfment enhances cell survival when cells are subjected to weak pro-apoptotic signals. Thus, genes that mediate corpse removal can also function to actively kill cells.

Hoeppner DJ et al. (1997) International C. elegans Meeting "TOWARD UNDERSTANDING CELL-TYPE SPECIFIC CELL DEATH REGULATION"

Hoeppner DJ, Desnoyers S, Hengartner MO

[International C. elegans Meeting, 1997]

Our goal is to understand how different cell types regulate programmed cell death in C. elegans. We begin by defining the genes necessary for Hermaphrodite-Specific Neuron (HSN) death. The HSNs are a pair of neurons that are required for egg-laying. The HSNs die by programmed cell death in males, presumably in response to masculine signals from the sex determination pathway. The HSNs also die in hermaphrodites carrying dominant gain-of function alleles of the egl-1 gene. Hermaphrodites lacking HSNs are defective in egg-laying (Egl), but are viable and display no pleiotropic defects. Thus, egl-1(gf) provides a good background to identify mutations in genes that are specifically required for HSN death, as restoration of HSN function must be achieved by blocking its death. To isolate egl-1(gf) suppressors we screened F1 and F2 progeny of mutagenized egl-1(n1084) animals for reversion of the egg-laying defect. In a screen of 100,000 haploid genomes, we isolated 34 suppressors, which fall into four classes: (I) loss-of-function alleles of ced-3 and ced-4, which block all cell deaths; (II) HSN-specific death suppressors; (III) bypass mutations that restore egg laying in the absence of HSNs and (IV) unstable suppressors of HSN death. Preliminary characterization of the HSN-specific suppressors will be presented.

Hoeppner DJ et al. (1998) East Coast Worm Meeting "CED-3 IS NOT ALONE: IDENTIFICATION OF TWO FURTHER CASPASE HOMOLOGS IN C. ELEGANS"

Lazebnik Y, Gartner A, Hoeppner DJ, Hengartner MO

[East Coast Worm Meeting, 1998]

C. elegans serves as a model system for elucidating the genetic basis of programmed cell death. Genetic analysis has provided evidence for a core apoptotic pathway in which the Bcl-2 homolog CED-9 regulates the activation of the effector caspase CED-3 via CED-4. Caspases are modular cysteine proteases with a regulatory amino-terminal prodomain and two catalytic carboxy-terminal subunits. In mammalian systems, some caspases act in sequential proteolytic cascades, where regulatory caspases respond to death signals and transduce these signals by proteolytic cleavage of effector caspases. In contrast to mammalian systems, CED-3 is the only caspase known to be involved in programmed cell death in C. elegans. We have recently cloned cDNAs for two further caspase homologs, csh-2 and csh-3. The catalytic residues are conserved between CED-3, CSH-2 and CSH-3, but some critical residues defining the substrate-binding pocket of CED-3 are not conserved. Further, the prodomains of CED-3, CSH-2 and CSH-3 are divergent, suggesting that these three proteases respond to different signals. Preliminary in vitro experiments indicate that CSH-2 has catalytic activity against synthetic tetrapeptide substrates. Extracts from E. coli expressing the CSH-2 catalytic domain, as a 6xHis-fusion protein in E. coli, prefer the tetrapeptide YVAD to DEVD. Considering that a large number of ced-3 alleles have been isolated in independent genetic screens, it is reasonable to propose that CED-3 is the only caspase required for programmed cell death in C. elegans. However, the presence of multiple caspases in C. elegans suggests that a death-inducing protease cascade may exist in nematodes. To explore this hypothesis, we have analyzed the cell death phenotype of a deletion allele of csh-2. This allele, csh-2(nr2018), prematurely truncates csh-2 upsteam of the catalytic domain. Worms homozygous for csh-2(nr2018) display no defects in programmed cell death. The cell death phenotypes in well-characterized cell death mutant backgrounds is neither enhanced nor suppressed by csh-2(nr2018), suggesting that csh-2 is either redundant, or not involved in cell death. We are presently generating a genomic deletion of the csh-3 locus to perform similar analyses and to address caspase redundancy in C. elegans.

Hoeppner DJ et al. (2004) Dev Biol "eor-1 and eor-2 are required for cell-specific apoptotic death in C. elegans."

Bhusri SS, Spector MS, Conradt B, Herman MA, Hoeppner DJ, Kinchen JM, Lin SC, Granat S, Ratliff TM, Hengartner MO

[Dev Biol, 2004]

Programmed cell death occurs in every multicellular organism and in diverse cell types yet the genetic controls that define which cells will live and which will die remain poorly understood. During development of the nematode Caenorhabditis elegans, the coordinated activity of four gene products, EGL-1, CED-9, CED-4 and CED-3, results in the death of essentially all cells fated to die. To identify novel upstream components of the cell death pathway, we performed a genetic screen for mutations that abolish the death of the hermaphrodite-specific neurons (HSNs), a homologous pair of cells required for egg-laying in the hermaphrodite. We identified and cloned the genes, eor-1 and eor-2, which are required to specify the fate of cell death in male HSNs. In addition to defects in HSN death, mutation of either gene leads to defects in coordinated movement, neuronal migration, male tail development, and viability; all consistent with abnormal neuronal differentiation. eor-1 encodes a putative transcription factor related to the human oncogene PLZF. eor-2 encodes a novel but conserved protein. We propose that eor-1 and eor-2 function together throughout the nervous system to promote terminal differentiation of neurons and function specifically in male HSNs to promote apoptotic death of the HSNs.

Clements CM et al. (2006) Proc Natl Acad Sci U S A "DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2."

McNally RS, Clements CM, Ting JP, Mak TW, Conti BJ

[Proc Natl Acad Sci U S A, 2006]

DJ-1/PARK7, a cancer- and Parkinson''s disease (PD)-associated protein, protects cells from toxic stresses. However, the functional basis of this protection has remained elusive. We found that loss of DJ-1 leads to deficits in NQO1 [NAD(P)H quinone oxidoreductase 1], a detoxification enzyme. This deficit is attributed to a loss of Nrf2 (nuclear factor erythroid 2-related factor), a master regulator of antioxidant transcriptional responses. DJ-1 stabilizes Nrf2 by preventing association with its inhibitor protein, Keap1, and Nrf2''s subsequent ubiquitination. Without intact DJ-1, Nrf2 protein is unstable, and transcriptional responses are thereby decreased both basally and after induction. This effect of DJ-1 on Nrf2 is present in both transformed lines and primary cells across human and mouse species. DJ-1''s effect on Nrf2 and subsequent effects on antioxidant responses may explain how DJ-1 affects the etiology of both cancer and PD, which are seemingly disparate disorders. Furthermore, this DJ-1/Nrf2 functional axis presents a therapeutic target in cancer treatment and justifies DJ-1 as a tumor biomarker.

Cornejo Castro EM et al. (2010) J Neural Transm "Parkinson's disease-associated DJ-1 modulates innate immunity signaling in Caenorhabditis elegans."

Autenrieth IB, Fiesel FC, Cornejo Castro EM, Schutz M, Oberhettinger P, Springer W, Kahle PJ, Waak J, Weber SS

[J Neural Transm, 2010]

DJ-1 is a neuroprotective gene mutated in recessive Parkinson's disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria, djr-1 (-/-) worms showed stronger phosphorylation of p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.

Lee JY et al. (2012) Hum Mol Genet "Human DJ-1 and its homologs are novel glyoxalases."

Lee JY, Song J, Kwon K, Park C, Baek K, Jang S, Kim C, Kim J

[Hum Mol Genet, 2012]

Human DJ-1 is a genetic cause of early-onset Parkinson's disease (PD), although its biochemical function is unknown. We report here that human DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid, respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit typical Michaelis-Menten kinetics, which were abolished completely in the mutants of essential catalytic residues, consisting of cysteine and glutamic acid. The presence of DJ-1 protected mouse embryonic fibroblast and dopaminergically derived SH-SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1, a DJ-1 homolog expressed primarily in the intestine, protected worms from glyoxal-induced death. Sub-lethal doses of glyoxals caused significant degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2, another DJ-1 homolog expressed primarily in the head region, including neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl species may provide a new insight into the causation of PD.

Johnson WM et al. (2016) Biochemistry "Regulation of DJ-1 by glutaredoxin 1 in vivo - implications for Parkinson's disease."

Mieyal JJ, Lin J, Currran PL, Wilson MA, Johnson WM, Ray A, Zhu X, Gorelenkova Miller O, Choe K, Yao C, Ravindranath V, Chen SG, Milkovic NM, Wang W, Golczak M, Wilson-Delfosse AL

[Biochemistry, 2016]

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH; and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in C. elegans, however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx-/- mice we provide evidence that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents; and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homolog in a C. elegans in vivo model of PD. Therefore; our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.