For human autosomal recessive diseases in which the responsible gene is known, we are using C. elegans to study the function of that gene and to genetically identify other factors that act in the same pathway. There are a number of criteria that must be met in order for this strategy to work. First, there must be a convincing and clear C. elegans ortholog. Second, there would have to be a mutation or deletion in this gene that already exists (alternatively, we could use the TALEN or CRISPR technology to generate mutant alleles). Third, there would have to be a scorable phenotype. The more penetrant the phenotype, the better. If these criteria are met, genetic suppressor and enhancer screens could be performed to identify interacting factors that function with any given gene and the biological process in which it functions. In the past year, we have identified a number of C. elegans orthologs of human disease-causing genes. We have determined that a number of these candidates satisfy all of the above criteria- there are mutations in these genes and they reveal very penetrant and scorable phenotypes. We are currently focusing on 2 type IV collagen genes, emb-9 and let-2, whose mutant phenotypes were previously characterized by Dr. James Kramer at Northwestern University. There are temperature-sensitive missense alleles of these genes, making them ideal for genetic suppression screens. We are also suppressing a ubiquitin activating enzyme, uba-1, which was originally identified and characterized by Dr. Harold Smith (Kulkarni and Smith, 2008), currently here in NIDDK. This allele is also temperature-sensitive. In addition to these genes, we have begun to characterize the germline defects of a deletion mutation in the ccm-3 ortholog of C. elegans;this is a gene that, when mutated, causes Cerebral Cavernous Malformations in humans. We are also characterized deletion alleles of sdhb-1, the succinate dehydrogenase gene and mev-1, a gene involved in oxidative phosphorylation. For each of these genes, mutations in them appear to reveal recessive highly penetrant phenotypes. We are carrying out straightforward genetic suppression screens with these mutants. The phenotypes of the above genes range from embryonic and larval lethality to slow development and sterility. The uba-1 mutant has a number of distinct phenotypes and thus we will need to score our suppressors for their ability to suppress each of these phenotypes. For diseases caused by dominant mutations, our strategy in the future will be different. We could attempt to express a dominant variant of the C. elegans ortholog. If there is a penetrant phenotype, we can characterize it and use genetic suppressor and enhancer screens to identify other factors that act in the same pathway. For genes that mutations do not exist, RNAi works extremely well and can also be used to screen for suppressors and enhancers to identify other factors interacting with a given gene. We have previously had success with a screen in which we suppressed an RNAi phenotype. However, with the CRISPR technology that has recently been shown to work very well in C. elegans, it will probably be easiest to generate small deletions or even specific missense mutations in genes of interest. With this technology, any gene can be mutated and tested for phenotypes, and then used in our genetic screens. We are currently testing the CRISPR technology to generate novel alleles of C. elegans genes. One clear example of this strategy came from the observation that an oncogenic ras mutant in C. elegans causes a Multivulva (Muv) phenotype. These Multivulva animals were subjected to suppressor screens in hopes of identifying extragenic mutations that suppress this dramatic phenotype. The ksr-1 gene was identified in these screens (Sundaram and Han, 1995;Kornfeld et al., 1995) and also in a Drosophila screen (Therrein et al., 1995);its name stands for kinase suppressor of ras. Loss-of-function alleles of ksr-1 suppress the dominant Multivulva phenotype of ras mutants in C. elegans. Mutations in any of the three human ras genes are thought to be present in at least 30% of all cancers. Specific mutations in K-Ras are thought to be present in 90% of all pancreatic cancers. Based on numerous studies in human cell lines, the inhibition or depletion of KSR1 is not detrimental to growth. In fact, the inhibition of KSR1 in mice bearing pancreatic tumor suggests that this might be a viable therapy for such tumors (Xing et al., 2003). Thus one could argue that the KSR gene would be a good therapeutic target for Ras-bearing mutations in humans as well. As demonstrated in the above example, drugs that mimic the effects of suppressor mutations might be worthy new therapies of diseases in which recessive mutations are known to be responsible for suppression. In the absence of effective gene therapy or stem cell therapy, drugs that target specific proteins may be beneficial to patients with such diseases.
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