Ghosh, Bhaswati et al. (2021) International Worm Meeting "A fat-promoting botanical extract from Artemisia scoparia acts as longevity modifier in C. elegans"

Guidry, Hayden, Bohnert, Adam, Ghosh, Bhaswati, Johnston, Maxwell

[International Worm Meeting, 2021]

Like other biological processes, aging is not random, but subject to molecular control. Natural products that act on conserved metabolic pathways may provide entry points to extend animal lifespan and promote healthy aging. Here, we show that a botanical extract from Artemisia scoparia (SCOPA), which has previously been reported to promote fat storage and metabolic resiliency in mice, exerts pro-longevity effects on the nematode Caenorhabditis elegans. We find that wild-type C. elegans treated with SCOPA show significantly higher fat levels than controls but live nearly 40% longer. The SCOPA-mediated changes to fat accumulation and lifespan require the transcription factor DAF-16/FOXO, which we find shuttles into the nucleus upon SCOPA treatment. Notably, the expression of DAF-16-targeted delta-9 desaturases dramatically increases in SCOPA-treated animals; these desaturases govern SCOPA's effects on fat content and are partly required for the SCOPA-dependent lifespan extension. We also find that SCOPA can enhance lifespan even when its administration is initiated at mid-adulthood, supporting its potential application as an intervention. In addition to their extended lifespan, SCOPA-treated animals show some signs of improved health during aging, including superior stress resistance. Thus, our data suggest that SCOPA-treated worms are fatty but metabolically healthy, and that they live long in part due to altered fat regulation. These findings add to emerging evidence indicating that elevated fat can be pro-health, and even pro-longevity, in some contexts.

Parker JA et al. (1997) International C. elegans Meeting "A C. elegans huntingtin interacting protein?"

Parker JA, Rose AM

[International C. elegans Meeting, 1997]

One of the areas of research in our lab concerns the identification and placement of lethal mutations along LGIII. The recent identification of a gene within this area has led to a collaborative effort between ourselves and the Michael Hayden lab, here at UBC. Within LGIII there is a gene that shares considerable sequence identity with human hip-1 (huntingtin interacting protein 1), and SLA-2 in yeast. Hip-1 has been found to interact with the Huntingtons disease gene product, huntingtin. (M. Kalchman, M. Hayden, personal communication). Hip-1s ability to interact with huntingtin is modulated by the size of the expanded polyglutamine repeat in the first exon of huntingtin. The repeat is greatly expanded in individuals with Huntingtons disease, and the strength of the hip-1/huntingtin interaction decreases with an increase in repeat size. Thus, the specific neuronal deterioration observed in Huntingtons disease may be attributable to the role of hip-1. Previous work has localised huntingtin to the plasma membrane.(Gutekunst,1995) This observation is supported by the observation that hip-1 shares identity with talin. Talin is a member of a group of proteins postulated to serve as structural links between the plasma membrane and the cytoskeleton. SLA-2 is a synthetic lethal mutation of yeast, and has been found to be membrane and cytoskeletal associated protein as well. Although, the yeast system is an excellent one for the study of the cellular function of this gene, C. elegans could provide insights into its potential developmental and tissue specificity. We have been characterising the 5 prime end of the gene in order to study its expression pattern. In addition, we are surveying our library of existing mutants for possible candidates, as well as conducting new mutagenesis screens.

Parker JA et al. (1999) Worm Breeder's Gazette "C. elegans HIP1 expression is restricted to a few neurons"

Parker JA, Rose AM

[Worm Breeder's Gazette, 1999]

We have been characterizing the expression of ZK370.3, which shares sequence identity with human HIP1 (Huntingtin Interacting Protein) and Sla2p of yeast (Synthetic lethal with Actin Binding protein 1). ZK370.3 spans approximately 4.5 kb of genomic DNA, consisting of 10 exons and codes for a 2.7 kb message. The 923 amino acid product has a molecular weight of approximately 100 kDa, and shares identity with cytoskeletal associated protein talin. To study the expression of C. elegans HIP1, 1000 nucleotides of the genes 5 upstream region, along with the first 30 nucleotides of the coding region, were fused to a GFP fusion vector (pPD95.75, kindly provided by A. Fire). The fusion construct was injected into dpy-5 animals, along with a dpy-5 rescuing clone (pCeh361, kindly provided by C. Thacker). Rescued animals were examined, and fluorescence was detected in the pharynx and tail of the animals. Expression in the pharynx appears to be limited to a set of neurons, possibly M4 and I3. To better understand the function of the C. elegans HIP1 gene product, we conducted a yeast two-hybrid screen in collaboration with the Michael Hayden laboratory at the University of British Columbia. We used a full length C. elegans HIP1 cDNA fused to the bait vector pGBT9 (Clonetech) to screen a library kindly provided by R. Barstead. Two clones were isolated from the screen. The first shows similarity to a type II transcription factor, and the second shows similarity to the Drosophila hedgehog protein. We are currently characterizing the protein interactions.

Ponce, Melissa et al. (2011) International Worm Meeting "A possible role for TRY-2/plasminogen during C. elegans embryonic development."

Ponce, Melissa, Sarraf-Mamouri, Saeideh, Howard, Austin, Hudson, Martin

[International Worm Meeting, 2011]

How neurons modify the extracellular matrix during nervous system development is poorly understood. Plasminogen-like proteases are required for cell migration and axon outgrowth of the dorsal root ganglia (1, 2) and are implicated in mossy fiber axon branching in the hippocampus (3). The use of proteolytic activity to facilitate axon outgrowth must be carefully controlled and coordinated with instructions from multiple axon guidance molecules. How does a neuron integrate these extracellular cues with protease activity to accurately permit axon growth through time and space? The kal-1/anosmin gene is implicated in multiple aspects of neuronal development including cell migration and axon branching in both vertebrates and invertebrates (4-7). In C. elegans, KAL-1 has high affinity for heparan sulfate proteoglycans (HSPGs); key components of the cell surface and extra cellular matrix. In addition, human anosmin exhibits high affinity binding to urokinin-related plasminogen activator (PLAU) in vitro. As such, KAL-1/anosmin is well positioned to integrate HSPG and extracellular matrix cues with the proteolytic activity of plasminogen activators, facilitating accurate axon outgrowth. PLAU contains a plasminogen protease and kringle domain. Our preliminary data indicates that C. elegans TRY-2 may be orthologous to human PLAU as this is the only C. elegans protease that contains both of these domains. In addition, try-2(ok2531) embryos exhibit neuroblast migration defects suggesting a role for plasminogen activity in embryonic development. Genetic analysis is ongoing to establish whether try-2 functions with other genes required for neuroblast migration, axon outgrowth and KAL-1 dependent axon branching. 1. Hayden and Seeds (1996) J Neurosci. 16, 2307-2317. 2. Hoover-Plow et al. (2001) Brain Res. 898, 256-264. 3. Wu et al. (2000) J Cell Bio. 148, 1295-1304. 4. Soussi-Yanicostas et al. (2002) Cell. 109, 217-228. 5. Bulow et al. (2002) Proc Natl Acad Sci USA. 99, 6346-6351. 6. Rugarli et al. (2002) Development. 129, 1283-1294. 7. Hudson et al. (2006) Dev Biol. 294, 352-365.

A Parker et al. (1999) International C. elegans Meeting "Studies of C. elegans HIP1"

A Parker, A Rose

[International C. elegans Meeting, 1999]

We are interested in determining the function of a gene in C. elegans , CeHIP1, which shares sequence identity with human HIP1 (Huntingtin Interacting Protein 1) and Sla2 of yeast. CeHIP1, HIP1, and Sla2 also share sequence identity with the C-terminus of vertebrate talin. Specifically, these proteins contain a conserved protein domain, known as an I/LWEQ module. This module has been shown to bind F-actin (McCann and Craig, PNAS, 1996). We wish to study CeHIP1's role in developmental and tissue specificity in C. elegans . We have characterized the gene structure of CeHIP1 and have determined it to be trans-spliced to SL1. We cloned the putative promoter region into a GFP expression vector. Fluorescence could be detected in pharyngeal neurons, possibly the M4 and I3 interneurons. CeHIP1 maps between dpy-19 and sma-2 on LGIII, and maps to cosmid ZK370 on the physical map. The genetic map indicates that nDf17 should uncover CeHIP1(ZK370.3) Using PCR, primers specific to CeHIP1 amplified DNA from nDf17 homozygotes. The control, primers for sma-2 (ZK370.4), do not amplify. Thus, it appears that nDF17 does delete DNA in the region corresponding to cosmid ZK370, but does not delete the CeHIP1 sequence tested (i.e. CeHIP1 appears to be skipped in nDf17 ). In addition, three deletion candidates have been identified from PCR screens of mutagenized libraries, but none of the deletion bearing animals could be recovered. We have used RNAi to study the effects of a reduction of CeHIP1. The progeny of injected animals have an increased rate of embryonic lethality in comparison to wild type. A number of unshelled oocytes were also present on the plates. We have conducted a yeast two-hybrid screen to identify interacting protein partners of CeHIP1. Two clones were isolated from the screen. The first shows identity to a transcription initiation factor type IIB. The second clone shows identity to the C-terminal portion of the Drosophila hedgehog protein. In collaboration with the Michael Hayden laboratory (University of British Columbia), we are characterizing nematode-human protein interactions. There is evidence that overexpression of HIP1 in human cells cause cell death through an apoptotic pathway (A. Hackam, personal communication). We are working to determine if overexpression of CeHIP1 causes a similar condition in C. elegans .

Neri C et al. (2000) European Worm Meeting "Modeling human disease neurodegenerative processes in C. elegans"

Holbert S, Connolly J, Chalfie M, Denghien I, Neri C

[European Worm Meeting, 2000]

The goal of our laboratory is to generate C. elegansmodels which are representative of different human disease neurodegenerative processes and suitable for the identification of new and potentially interesting therapeutic targets. Our approach relies on the combined use of transgenesis in worms and two-hybrid selection in yeast. Our research is currently focused on attempting to understand the bases of Huntingtons Disease (HD). This is one of the most common neurological disorders where expanded tracts of polyglutamine in the disease-associated protein are correlated directly with the severity of symptoms and inversely with the age-of-onset. In collaboration with Columbia University, we have used the promoter from the mec3 gene to drive transgene expression in the six touch receptor neurons, which underlie response to gentle touch in worms. Transgenics expressing GFP fused to the first 57 N-terminal amino acids of human huntingtin (htt) containing normal and expanded polyglutamine tracts show polyglutamine length-dependent defective neuronal phenotypes. In particular, tail mechanosensation, mediated by the PLM neurons, is compromised. We observe aggregate formation appearing in a polyglutamine length-dependent fashion. However, we do not observe significant formation of nuclear inclusions. In addition to screening for suppression of transgenic phenotypes induced by expanded polyglutamine tracts, we are currently carrying out a detailed cell biological analysis of PLM neurons in transgenics expressing polyglutamines, as well as continuing to assess the effects of expanded polyglutamines in other neuronal cell types (*). In parallel, we have screened a worm cDNA library (R. Barstead, OKMRF, Oklahoma City, OK) by using two-hybrid selection in yeast. We used htt546Q15 as a bait-protein, a N-terminal huntingtin fragment that contains amino acids 1-546 with 15 consecutive glutamines, and other constructs expressing htt in post-screening studies (C. Wellington and M. Hayden, CMMT, University of British Columbia, Vancouver, Canada). We have identified a protein (ZK1127.9) able to interact with htt546Q15. ZK1127.9 shows a loss of interaction with htt546Q128. ZK1127.9 is homologous with a human nuclear protein (CA150), both proteins showing three predicted WW domains. As compared to ZK1127.9, the N-terminal region of CA150 shows a polypeptide insertion that contains a (QA)38 repeat found to be polymorphic in CEPH pedigrees (apparent heterozygosity: 30%). The CA150 protein is a putative transcription factor associated with the RNA polymerase II holoenzyme (Sune et al., 1997, Mol. Cell. Biol. 17: 60 29-6039) that may be expressed in the brain as suggested by the presence of several human brain ESTs in Genbank. We are currently testing whether htt might interact with CA150. Should mutated htt fragments be revealed to interact inappropriately with CA150, CAG expansion in the IT15 gene and subsequent variation in the expression of genes essential to neuron function might participate to the etiology of HD. In conjunction with this notion, the polymorphic (QA)38 repeat in CA150 might modulate the interaction of this protein with htt and have a role in the progression of HD. Based on the evidence for interaction of huntingtin and polyglutamine tracts with cellular mechanisms in C. elegans, our approach is being applied to the analysis of neurodegenerative processes involved in human diseases other than HD. (*) supported by the Hereditary Disease Foundation Cure HD Initiative.