Jae Hyung An et al. (2004) East Coast Worm Meeting "Regulation of a Conserved Oxidative Stress Defense by GSK-3 and p38 signaling in C. elegans"

T Keith Blackwell, Rosanna Baker, Kelly Vranas, Hideki Inoue, Yanxia Bei, Naoki Hisamoto, Craig C Mello, Jae Hyung An, Kunihiro Matsumoto, Riva Oliveira

[East Coast Worm Meeting, 2004]

Oxidative stress is a central etiologic factor in diabetes, cardiovascular disease, reperfusion injury, and various other pathologies. In vertebrates a major defense against oxidative and xenobiotic stress is orchestrated by the two Nrf bZIP transcription factors, which induce expression of a battery of Phase II detoxification enzymes. These enzymes synthesize glutathione and scavenge free radicals directly. This stress response system is also activated by chemoprotective antioxidants that are produced by many plants, and can inhibit chemical carcinogenesis in mice. We have determined that in C. elegans , this oxidative stress defense is mediated by the transcription factor SKN-1 (An and Blackwell (2003) Genes Dev., 17, 1882). SKN-1 is distantly related to Nrf proteins but binds DNA through a unique monomeric mechanism, indicating that this detoxification system has been conserved despite a dramatic divergence in DNA recognition. Previous work showed that in the embryo, maternally expressed SKN-1 initiates formation of the entire digestive system and other mesendodermal tissues. We have found that during postembryonic stages, zygotically expressed SKN-1 accumulates in intestinal nuclei in response to oxidative stress and directly regulates a key Phase II gene through constitutive and stress-inducible mechanisms in different tissues. In contrast, SKN-1 is constitutively nuclear and active in the ASI neurons. skn-1 mutants are sensitive to oxidative stress, and age prematurely. We believe that the developmental function of SKN-1 arose from this ancient conserved stress response. We are now investigating how this detoxification response is regulated in the C. elegans intestine. The Matsumoto lab has shown that a p38-like stress-activated kinase (PMK-1) phosphorylates SKN-1, and p38 signaling is required for SKN-1 to accumulate in nuclei or activate target genes in response to oxidative stresses. We have found that the C. elegans glycogen synthase kinase-3 (GSK-3) alpha/beta ortholog gsk-3 prevents constitutive SKN-1 activation. When gsk-3 is inhibited by RNAi, SKN-1 is localized to intestinal nuclei without oxidative stress. A direct GSK-3 phosphorylation site and putative priming kinase site that we have identified are each required to prevent constitutive localization of SKN-1 to intestinal nuclei. The p38 pathway regulates SKN-1 epistatically to GSK-3, indicating that it is required for SKN-1 function independently of mechanisms that overcome effects of GSK-3 on SKN-1. Our experiments have identified novel components of this conserved oxidative stress response, and have shown that oxidative stress resistance is modulated by a kinase (GSK-3) that has many key functions in metabolism, growth control, and differentiation. We believe that in the intestine SKN-1 integrates multiple redox and metabolic inputs. Mechanisms through which these and other signals may regulate SKN-1 are under investigation.

Kathleen Estes et al. (2007) International Worm Meeting "C. elegans vulva development as a model for determining the role of UNC-6 Netrin in tissue morphogenesis."

Kathleen Estes, Wendy Hanna-Rose

[International Worm Meeting, 2007]

UNC-6 Netrin is a well-studied axon guidance molecule. Recently, this protein has been observed acting in morphogenesis of different tissues1. C. elegans unc-6 mutants have a well documented egg-laying defect2. Interestingly, a vulva morphology defect has also been reported but not well-characterized2. We are currently conducting an in-depth phenotypic analysis of the vulva morphology defect in various unc-6 alleles. We are also examining unc-40, the only one of the two well-characterized unc-6 receptors to exhibit an egg-laying defect2. In our initial analysis using an apical junction marker, we have found that the dorsal vulva toroid of unc-6 mutants is severely misshapen and does not seem to display this marker. We also have preliminary data suggesting the anchor cell may not invade the basement membrane, which may contribute to the vulva morphology defect. By studying the phenotype of unc-6 and unc-40 mutants in C. elegans vulva morphogenesis, we can examine this new role of netrin in tissue morphogenesis. 1. reviewed in Baker, et. al. 2006. Curr Opin Neurobiol 16: 529-534 2. Hedgecock, et. al. 1990. Neuron 2: 61-85.

Kao, Aimee Wen Yi et al. (2009) International Worm Meeting "Loss of the C. elegans progranulin homolog, PGRN-1, results in decreased programmed cell death and an age-dependent response to cellular stress."

Farese, Robert, Bagley, Joshua, Kao, Aimee Wen Yi, Kenyon, Cynthia, Nakamura, Ayumi, Eisenhut, Robin

[International Worm Meeting, 2009]

Frontotemporal lobar degeneration (FTLD) is the second most common cause of dementia in those under the age of 65. Mutations in the human progranulin (PGRN) gene have recently been shown to be causal for both inherited and sporadic forms of FTLD (Baker et al., 2006, Hutton et al., 2006). Progranulin is a complex, multifunctional, secreted trophic factor that is expressed in a variety of tissues including neurons and astrocytes (Ahmed et al., 2007). Although PGRN is involved in development, wound healing, inflammation and tumor growth, its function in the nervous system is poorly understood (Eriksen and Mackenzie, 2008). In order to better understand the biological function of progranulin, we decided to study its homolog in C. elegans. Similar to the mammalian protein, the C. elegans progranulin homolog, PGRN-1, is expressed by the intestine and a subset of neurons. pgrn-1 mutant worms appear grossly normal and live a normal lifespan compared to control worms. However, we have found that pgrn-1 mutants have a decreased number of programmed cell deaths during development. In addition, young adult pgrn-1 mutants display resistance to osmotic, thermal and ER stress compared to wild-type worms while older worms are sensitive to osmotic stress. The stress resistance phenotype can be rescued by expression of either worm or human progranulin. We are currently examining whether these constructs also rescue the cell death phenotype. Unlike mutations in the insulin-IGF-1 receptor gene, daf-2, pgrn-1 mutations do not cause resistance to genotoxic or oxidative stress, suggesting a novel pathway for selective stress resistance. This dichotomous response to certain stressors-resistance while young and increased susceptibility with age-may shed light on why individuals with progranulin mutations develop FTLD symptoms late in adulthood and may explain the variable age of disease onset seen with identical mutations. Interestingly, our findings suggest that susceptibility to neurodegeneration may be determined during a developmental period when excess or unneeded neurons undergo programmed cell death.

Wang, A. et al. (2019) International Worm Meeting "Progranulin Polymorphisms in Aging and Stress Resistance."

Kao, A, Cortopassi, W, Butler, V, Jacobson, M, Wang, A.

[International Worm Meeting, 2019]

The cysteine-rich protein progranulin (PGRN) has been implicated in the pathobiology of several diseases including neurodegeneration and lysosomal storage related diseases (Baker, Nature 2006; Cruts, Nature 2006; Neumann, Science 2006; Smith, Am J Hum Genet 2012). Progranulin can be processed into bioactive granulin peptides, the number of which vary from species to species. C. elegans progranulin contains 3 granulins. Despite the variance in number of domains, the granulins share a stable, compact structure of beta-pleated sheets connected by disulfide bonds(Palfree, Plos One 2015). Studies have shown that loss of progranulin leads to features of accelerated aging, such as lipofuscin deposits and decreased lysosome protease activity (Ahmed, The American Journal of Pathology 2010; Ward, Science Translational Medicine 2017). In killifish, progranulin polymorphisms segregate with a short-lived aging phenotype (H151Q, C449W, R158H) (Valenzano, Cell 2015). The cysteine to tryptophan polymorphism is particularly interesting due to the cysteine's structural contribution to disulfide bonds. In addition, the histidine to glutamine polymorphism could potentially alter pH sensitivity. We have shown that pgrn-1(-) animals demonstrate a robust, stress-resistant phenotype that interestingly, can be decoupled from lifespan extension (Butler, J Neurosci 2019; Meredith, Plos Genetics 2013). Furthermore, progranulin acts in the lysosome and directly increases the activity of cathepsin-D (CTSD), a crucial lysosomal protease (Butler, Human Molecular Genetics 2018). We hypothesize that while complete absence of progranulin and granulin does not affect lifespan, that the polymorphisms in the granulin domains will impact aging and lifespan as well as stress response via regulation of lysosomal protease activity. To address this, we have created single copy insertions using MosSCI of these progranulin SNPs into C. elegans. We plan to test lifespan of these worms. We will also measure markers of aging and test stress response. To better understand potential molecular mechanisms, we will use molecular modeling to elucidate changes to progranulin and CTSD/ASP-3 interactions and test these in vivo. We anticipate that certain progranulin polymorphisms will increase lifespan and healthspan. We also predict that stress resistance will improve. We anticipate these will be a result of progranulin polymorphisms enhancing proteostasis via improved lysosomal protease activity.

Stephen D Fields et al. (2003) International Worm Meeting "HUM-2, a myosin V homologue, is expressed in head neurons and affects the basal slowing response to food."

John R McManus, Stephen D Fields, Jim Rand, Greg Mullen

[International Worm Meeting, 2003]

Myosin V, an unconventional myosin, seems to be common to most organisms but takes part in rather diverse processes, including localization of mRNA, recycling plasma membrane receptors, and positioning melanosomes and synaptic vesicles. Myosin V is expressed in mammalian nervous systems, and mutations in human myosin V are associated with at least one neurological disease. Accordingly, we are interested in whether HUM-2, the C. elegans homologue of myosin V (Baker and Titus, 1997), is expressed in neurons. HUM-2 is predicted to have several alternatively spliced variants, the longest of which is HUM-2b. We performed 5' RACE for the hum-2b transcript and found that the actual start codon does not correspond to the site predicted by genefinder. We used the PCR-based method of Hobert et al (2002) to fuse the upstream promoter region and first exon of hum-2b to GFP. This product, along with pBX, was injected into Pha-1 worms, and seven GFP-expressing transgenics were obtained. Most of the transgenic lines express GFP in six to eight head neurons with cell bodies in the anterior and lateral ganglia. At least four of these neurons send processes to the nose as well as into the nerve ring and occasionally into the ventral nerve cord. Those worms showing the strongest expression also have one or two GFP-positive cell bodies in the pre-anal ganglion, as well as the anal sphincter muscle. In addition, all lines express the promoter driven GFP in the pharyngeal-intestinal valve, and several lines weakly express GFP in the excretory system. The C. elegans knockout consortium has isolated a 2.1 kb deletion in the hum-2 gene (OK596) that begins in the region corresponding to the motor domain of the predicted protein. This deletion is expected to result in a truncated, null protein. The outcrossed hum-2 mutants do not have an obvious phenotype although they do not exhibit the typical slowing response upon entering food, suggesting that there is either a mechano- or chemosensory defect. We are in the process of identifying the relevant neurons, but the GFP expression pattern and the modulation defect in the mutant suggets that the dopaminergic neurons are involved. Finally, we are determining the expression pattern of the predicted splice variants of HUM-2, including HUM-2c, which is a truncated version of HUM-2b lacking the motor domain.