Environmental carcinogen benzo[a]pyrene alters neutral lipid storage via a cyp-35A2 mediated pathway in Caenorhabditis elegans.

Polycyclic aromatic hydrocarbons (PAHs), in particular benzo [a]pyrene (BaP), have been identified as carcinogenic components of tobacco smoke. In mammals, the toxicological response to BaP-diol-epoxide is driven by cytochrome P450 (CYP1A1), a pathway which is absent in Caenorhabditis elegans. In contrast, in worms prominently the CYP-35 enzyme family seems to be induced after BaP exposure. In C. elegans, BaP exposure reduces the accumulation of lysosomal neutral lipids in a dose dependent manner and the deletion of cyp-35A2 results in a significant elevation of neutral lipid metabolism. A cyp-35A2:mCherry;unc-47:GFP dual-labelled reporter strain facilitated the identification of three potential upstream regulators that drive BaP metabolism in worms, namely elt-2, nhr-49 and fos-1. This newly described reporter line is a powerful resource for future large-scale RNAi regarding toxicology and lipid metabolism screens.

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA.

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lag behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development.

A humanized Caenorhabditis elegans model of hereditary spastic paraplegia-associated variants in KLC4.

Hereditary spastic paraplegia (HSP) is a group of degenerative neurological disorders. We identified a variant in human kinesin light chain 4 (KLC4) that is suspected to be associated with autosomal-dominant HSP. How this and other variants relate to pathologies is unknown. We created a humanized Caenorhabditis elegans model in which klc-2 was replaced by human KLC4 (referred to as hKLC4) and assessed the extent to which hKLC4 retained function in the worm. We observed a slight decrease in motility but no nuclear migration defects in the humanized worms, suggesting that hKLC4 retains much of the function of klc-2. Five hKLC4 variants were introduced into the humanized model. The clinical variant led to early lethality, with significant defects in nuclear migration when homozygous and a weak nuclear migration defect when heterozygous, possibly correlating with the clinical finding of late-onset HSP when the proband was heterozygous. Thus, we were able to establish humanized C. elegans as an animal model for HSP and to use it to test the significance of five variants of uncertain significance in the human gene KLC4.

Generation of a C. elegans tdp-1 null allele and humanized TARDBP containing human disease-variants.

Clinical variants of TARDBP are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and other degenerative diseases. The predicted C. elegans ortholog of TARDBP is encoded by tdp-1 , but functional orthology has not been demonstrated in vivo. We undertook CRISPR/Cas9-based genome editing of the tdp-1 locus to create a complete loss of function allele; all tdp-1 exons and introns were deleted, creating tdp-1(tgx58) , which resulted in neurodegeneration after oxidative stress. Next, we undertook CRISPR-based genome editing to replace tdp-1 exons with human TARDBP coding sequences, creating humanized ( hTARDBP ) C. elegans expressing TDP-43 . Based on the efficiency of this genome editing, we suggest that iterative genome editing of the tdp-1 target locus using linked coCRISPR markers, like dpy-10 , would be a more efficient strategy for sequential assembly of the large engineered transgenes. hTARDBP decreased the neurodegeneration defect of tdp-1(tgx58) , demonstrating functional cross-species orthology. To develop C. elegans models of FTD and ALS, we inserted five different patient TARDBP variants in the C. elegans hTARDBP locus. Only one clinical variant increased stress-induced neurodegeneration; other variants caused inconsistent or negligible defects under these conditions. Combined, this work yielded an unambiguous null allele for tdp-1 , a validated, humanized hTARDBP, and multiple ALS/FTD patient-associated variant models that can be used for future studies.

A humanized Caenorhabditis elegans model of Hereditary Spastic Paraplegia-associated variants in kinesin light chain KLC4.

Hereditary spastic paraplegia (HSP) is a group of degenerative neurological disorders. We identified a variant in human kinesin light chain KLC4 that is suspected to be associated with autosomal dominant HSP. How this and other variants relate to pathologies is unknown. We created a humanized C. elegans model where klc- 2 was replaced with human KLC4 and assessed the extent to which hKLC4 retained function in the worm. We observed a slight decrease in motility but no nuclear migration defects in the humanized worms, suggesting that hKLC4 retains much of the function of klc-2 . Five hKLC4 variants were introduced into the humanized model. The clinical variant led to early lethality with significant defects in nuclear migration when homozygous, and a weak nuclear migration defect when heterozygous, possibly correlating with the clinical finding of late onset HSP when the proband was heterozygous. Thus, we were able to establish humanized C. elegans as an animal model for HSP and use it to test the significance of five variants of uncertain significance in the human gene KLC4 . Summary Statement: We identified a variant in KLC4 associated with Hereditary Spastic Paraplegia. The variant had physiological relevance in a humanized C. elegans model where we replaced klc-2 with human KLC4 .

Phenotypic screening models for rapid diagnosis of genetic variants and discovery of personalized therapeutics.

Precision medicine strives for highly individualized treatments for disease under the notion that each individual's unique genetic makeup and environmental exposures imprints upon them not only a disposition to illness, but also an optimal therapeutic approach. In the realm of rare disorders, genetic predisposition is often the predominant mechanism driving disease presentation. For such, mostly, monogenic disorders, a causal gene to phenotype association is likely. As a result, it becomes important to query the patient's genome for the presence of pathogenic variations that are likely to cause the disease. Determining whether a variant is pathogenic or not is critical to these analyses and can be challenging, as many disease-causing variants are novel and, ergo, have no available functional data to help categorize them. This problem is exacerbated by the need for rapid evaluation of pathogenicity, since many genetic diseases present in young children who will experience increased morbidity and mortality without rapid diagnosis and therapeutics. Here, we discuss the utility of animal models, with a focus mainly on C. elegans, as a contrast to tissue culture and in silico approaches, with emphasis on how these systems are used in determining pathogenicity of variants with uncertain significance and then used to screen for novel therapeutics.

The use of CRISPR to generate a whole-gene humanized MAPT and the examination of P301L and G272V clinical variants, along with the creation of deletion null alleles of ptl-1, pgrn-1 and alfa-1 loci.

To study important genes involved in Frontotemporal Dementia ( MAPT , GRN and C9orf72 ), we created deletion alleles in the three orthologous genes ( ptl-1 , pgrn-1 , and alfa-1 ). Simultaneously, we replaced the C. elegans ptl-1 gene with the predicted orthologous human MAPT gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an unc-18 mutation rescue technique. Clinical MAPT ALS/FTD missense variants G272V and P301L were successfully inserted in hMAPT . Neither ptl-1 loss or clinical variants caused neuronal defects in young adult or aged C. elegans , based on examination of glutamatergic phasmid neurons. Yet, we noted decreased survival to day 9 in the P301L hMAPT strain, compared to control strains. Based on these results, we comment on strategies for humanization, including the importance of confirming C. elegans gene predictions and identifying loss of function defects for each gene before embarking on humanization, and we report the creation of strains and a new gene-editing selection strategy that will be useful for future studies.

Atropine reduces aldicarb-induced sensitivity to C. elegans electroshock model.

Atropine has been used as an established anticonvulsant treatment for nerve agent intoxication. Atropine reduces electroshock recovery time among aldicarb-exposed wild-type C. elegans .

Neuronal control of maternal provisioning in response to social cues.

Mothers contribute cytoplasmic components to their progeny in a process called maternal provisioning. Provisioning is influenced by the parental environment, but the molecular pathways that transmit environmental cues between generations are not well understood. Here, we show that, in Caenorhabditis elegans, social cues modulate maternal provisioning to regulate gene silencing in offspring. Intergenerational signal transmission depends on a pheromone-sensing neuron and neuronal FMRFamide (Phe-Met-Arg-Phe)-like peptides. Parental FMRFamide-like peptide signaling dampens oxidative stress resistance and promotes the deposition of mRNAs for translational components in progeny, which, in turn, reduces gene silencing. This study identifies a previously unknown pathway for intergenerational communication that links neuronal responses to maternal provisioning. We suggest that loss of social cues in the parental environment represents an adverse environment that stimulates stress responses across generations.

A Caenorhabditis elegans model for ether lipid biosynthesis and function.

Ether lipids are widespread in nature, and they are structurally and functionally important components of membranes. The roundworm, Caenorhabditis elegans, synthesizes numerous lipid species containing alkyl and alkenyl ether bonds. We isolated C. elegans strains carrying loss-of-function mutations in three genes encoding the proteins required for the initial three steps in the ether lipid biosynthetic pathway, FARD-1/FAR1, ACL-7/GNPAT, and ADS-1/AGPS. Analysis of the mutant strains show that they lack ether lipids, but possess the ability to alter their lipid composition in response to lack of ether lipids. We found that increases in de novo fatty acid synthesis and reduction of stearoyl- and palmitoyl-CoA desaturase activity, processes that are at least partially regulated transcriptionally, mediate the altered lipid composition in ether lipid-deficient mutants. Phenotypic analysis demonstrated the importance of ether lipids for optimal fertility, lifespan, survival at cold temperatures, and resistance to oxidative stress.Caenorhabditis.

Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.

Fatty acid desaturation and the regulation of adiposity in Caenorhabditis elegans.

Monounsaturated fatty acids are essential components of membrane and storage lipids. Their synthesis depends on the conversion of saturated fatty acids to unsaturated fatty acids by Delta9 desaturases. Caenorhabditis elegans has three Delta9 desaturases encoded by the genes fat-5, fat-6, and fat-7. We generated nematodes that display a range of altered fatty acid compositions by constructing double-mutant strains that combine mutations in fat-5, fat-6, and fat-7. All three double-mutant combinations have reduced survival at low temperatures. The fat-5;fat-6 double mutants display relatively subtle fatty acid composition alterations under standard conditions, but extreme fatty acid composition changes and reduced survival in the absence of food. The strain with the most severe defect in the production of unsaturated fatty acids, fat-6;fat-7, exhibits slow growth and reduced fertility. Strikingly, the fat-6;fat-7 double-mutant animals have decreased fat stores and increased expression of genes involved in fatty acid oxidation. We conclude that the Delta9 desaturases, in addition to synthesizing unsaturated fatty acids for properly functioning membranes, play key roles in lipid partitioning and in the regulation of fat storage.

Genetic regulation of unsaturated fatty acid composition in C. elegans.

Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor, NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex regulation of the delta-9 desaturases to achieve optimal fatty acid composition.