Mutations in Caenorhabditis elegans neuroligin-like glit-1, the apoptosis pathway and the calcium chaperone crt-1 increase dopaminergic neurodegeneration after 6-OHDA treatment.

The loss of dopaminergic neurons is a hallmark of Parkinson's disease, the aetiology of which is associated with increased levels of oxidative stress. We used C. elegans to screen for genes that protect dopaminergic neurons against oxidative stress and isolated glit-1 (gliotactin (Drosophila neuroligin-like) homologue). Loss of the C. elegans neuroligin-like glit-1 causes increased dopaminergic neurodegeneration after treatment with 6-hydroxydopamine (6-OHDA), an oxidative-stress inducing drug that is specifically taken up into dopaminergic neurons. Furthermore, glit-1 mutants exhibit increased sensitivity to oxidative stress induced by H2O2 and paraquat. We provide evidence that GLIT-1 acts in the same genetic pathway as the previously identified tetraspanin TSP-17. After exposure to 6-OHDA and paraquat, glit-1 and tsp-17 mutants show almost identical, non-additive hypersensitivity phenotypes and exhibit highly increased induction of oxidative stress reporters. TSP-17 and GLIT-1 are both expressed in dopaminergic neurons. In addition, the neuroligin-like GLIT-1 is expressed in pharynx, intestine and several unidentified cells in the head. GLIT-1 is homologous, but not orthologous to neuroligins, transmembrane proteins required for the function of synapses. The Drosophila GLIT-1 homologue Gliotactin in contrast is required for epithelial junction formation. We report that GLIT-1 likely acts in multiple tissues to protect against 6-OHDA, and that the epithelial barrier of C. elegans glit-1 mutants does not appear to be compromised. We further describe that hyperactivation of the SKN-1 oxidative stress response pathway alleviates 6-OHDA-induced neurodegeneration. In addition, we find that mutations in the canonical apoptosis pathway and the calcium chaperone crt-1 cause increased 6-OHDA-induced dopaminergic neuron loss. In summary, we report that the neuroligin-like GLIT-1, the canonical apoptosis pathway and the calreticulin CRT-1 are required to prevent 6-OHDA-induced dopaminergic neurodegeneration.

Removal of extracellular human amyloid beta aggregates by extracellular proteases in C. elegans.

The amyloid beta (Aß) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aß aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo, we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aß1-42. This secreted Aß forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aß aggregation regulators, we identified different collagens to ameliorate or potentiate Aß aggregation. We show that a disintegrin and metalloprotease a disintegrin and metalloprotease 2 (ADM-2), an ortholog of ADAM9, reduces the load of extracellular Aß aggregates. ADM-2 is required and sufficient to remove the extracellular Aß aggregates. Thus, we provide in vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aß aggregate removal.

Youthful and age-related matreotypes predict drugs promoting longevity.

The identification and validation of drugs that promote health during aging ("geroprotectors") are key to the retardation or prevention of chronic age-related diseases. Here, we found that most of the established pro-longevity compounds shown to extend lifespan in model organisms also alter extracellular matrix gene expression (i.e., matrisome) in human cell lines. To harness this observation, we used age-stratified human transcriptomes to define the age-related matreotype, which represents the matrisome gene expression pattern associated with age. Using a "youthful" matreotype, we screened in silico for geroprotective drug candidates. To validate drug candidates, we developed a novel tool using prolonged collagen expression as a non-invasive and in-vivo surrogate marker for Caenorhabditis elegans longevity. With this reporter, we were able to eliminate false-positive drug candidates and determine the appropriate dose for extending the lifespan of C. elegans. We improved drug uptake for one of our predicted compounds, genistein, and reconciled previous contradictory reports of its effects on longevity. We identified and validated new compounds, tretinoin, chondroitin sulfate, and hyaluronic acid, for their ability to restore age-related decline of collagen homeostasis and increase lifespan. Thus, our innovative drug screening approach-employing extracellular matrix homeostasis-facilitates the discovery of pharmacological interventions promoting healthy aging.

Combining Auxin-Induced Degradation and RNAi Screening Identifies Novel Genes Involved in Lipid Bilayer Stress Sensing in Caenorhabditis elegans.

Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. We performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi in C. elegans to identify members of the lipid bilayer stress response. AID-mediated degradation of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, induced the activation of lipid bilayer stress-sensitive reporters. We screened through most C. elegans kinases and transcription factors by feeding RNAi. We discovered nine genes that suppressed the lipid bilayer stress response in C. elegans These suppressor genes included drl-1/MAP3K3, gsk-3/GSK3, let-607/CREB3, ire-1/IRE1, and skn-1/NRF1,2,3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Thus, we demonstrated proof-of-concept for combining AID and RNAi as a new screening strategy and identified eight conserved genes that had not previously been implicated in the lipid bilayer stress response.

The in-silico characterization of the Caenorhabditis elegans matrisome and proposal of a novel collagen classification.

Proteins are the building blocks of life. While proteins and their localization within cells and sub-cellular compartments are well defined, the proteins predicted to be secreted to form the extracellular matrix - or matrisome - remain elusive in the model organism C. elegans. Here, we used a bioinformatic approach combining gene orthology and protein structure analysis and an extensive curation of the literature to define the C. elegans matrisome. Similar to the human genome, we found that 719 out of ~20,000 genes (~4%) of the C. elegans genome encodes matrisome proteins, including 181 collagens, 35 glycoproteins, 10 proteoglycans, and 493 matrisome-associated proteins. We report that 173 out of the 181 collagen genes are unique to nematodes and are predicted to encode cuticular collagens, which we are proposing to group into five clusters. To facilitate the use of our lists and classification by the scientific community, we developed an automated annotation tool to identify ECM components in large datasets. We also established a novel database of all C. elegans collagens (CeColDB). Last, we provide examples of how the newly defined C. elegans matrisome can be used for annotations and gene ontology analyses of transcriptomic, proteomic, and RNAi screening data. Because C. elegans is a widely used model organism for high throughput genetic and drug screens, and to study biological and pathological processes, the conserved matrisome genes may aid in identifying potential drug targets. In addition, the nematode-specific matrisome may be exploited for targeting parasitic infection of man and crops.