Benbow, S.J. et al. (2017) International Worm Meeting "Characterization of Alzheimer's disease risk factors in a C. elegans model of tauopathy."

Benbow, S.J., Kraemer, B.C.

[International Worm Meeting, 2017]

Aberrant aggregation, misfolding, and mislocalization of the microtubule-associated protein tau characterize several neurodegenerative diseases, termed tauopathies. Alzheimer's disease (AD) is the most common tauopathy and most prevalent form of dementia within the global aging population. AD is distinguished from related dementias by the presence of two aggregant protein pathologies: intracellular neurofibrillary tangles (NFTs) composed of aggregated tau protein and extracellular amyloid- beta (A beta ) plaques. While heritable mutations in amyloid precursor protein (APP) and presenilins 1 and 2 (PSEN1, PSEN2) have considerably advanced our understanding of AD pathogenesis, they account for only a small portion of total AD cases. The majority of cases are classified as sporadic late-onset Alzheimer's disease and present without known causative mutations. However, human genomic studies have revealed a number of genetic variants contributing significant risk for AD. Genetic analysis by genome wide association studies (GWAS) has led to the identification of several novel genetic risk factors for AD (e.g. ABCA7, PICALM, DSG2, INPP5D, MEF2C, PTK2B, SLC24H4-RIN3, and ABI3). We hypothesize that a subset of these gene variants may modulate the severity of tau pathology, thereby contributing to the risk of developing AD. To test this hypothesis, we will explore whether these genes affect tau pathology in our C. elegans model of tauopathy. In this model, pan-neuronal expression of human tau recapitulates several features of human disease including accumulation of detergent-insoluble phosphorylated tau aggregates, abnormal behavior, neurodegeneration, and shortened lifespan. Tau transgenic C. elegans will be crossed with worms carrying a loss-of-function mutation in one of the homologous genes corresponding to a novel AD risk factor: abt-2, unc-11, hmr-1, unc-26, mef-2, kin-32, rin-1 and abi-1. Using behavioral analysis and biochemical assays to examine tau phosphorylation and aggregation, we will assess the impact of AD genetic risk factor genes on the severity of tau pathology. Identification of the molecular mechanisms underpinning genetic risk for AD remains a key unaddressed area and may provide novel therapeutic targets.

Benbow, S.J. et al. (2019) International Worm Meeting "Characterization of Alzheimer's disease risk factors in a C. elegans model of tauopathy."

Kraemer, B.C., Benbow, S.J.

[International Worm Meeting, 2019]

Aberrant aggregation, misfolding, and mislocalization of the microtubule-associated protein tau characterize several neurodegenerative diseases, termed tauopathies. Alzheimer's disease (AD) is the most common tauopathy and most prevalent form of dementia within the global aging population. AD is distinguished from related dementias by the presence of two aggregant protein pathologies: intracellular neurofibrillary tangles (NFTs) composed of aggregated tau protein and extracellular amyloid- beta (A beta ) plaques. While heritable mutations in amyloid precursor protein (APP) and presenilins 1 and 2 (PSEN1, PSEN2) have considerably advanced our understanding of AD pathogenesis, they account for only a small portion of total AD cases. The majority of AD cases present without known causative mutations. However, human genomic studies have revealed a number of genetic variants contributing significant risk for AD. Genetic analysis by genome wide association studies (GWAS) has led to the identification of several novel genetic risk factors (e.g. ABCA7, PICALM, DSG2, INPP5D, MEF2C, PTK2?, SLC24H4-RIN3, SYK and ABI3). We have observed that a subset of these gene variants modulate the severity of tau pathology in our C. elegans model of tauopathy. In this model, pan-neuronal expression of human tau recapitulates several features of human disease including accumulation of detergent-insoluble phosphorylated tau aggregates, abnormal behavior, neurodegeneration, and shortened lifespan. Loss of function mutations in C. elegans homologs corresponding to GWAS nominated genes abl-1 (SYK) and hmr-1 (DSG2) enhance motility defects in human tau expressing worms, while abt-2 (ABCA7) and kin-32 (PTK2?) do not. Candidate genes that affect tau-induced motility phenotypes will be further tested for the ability to modulate pathological tau and neurodegeneration. Identification of the molecular mechanisms underpinning genetic risk for AD remains a key unaddressed area and may provide novel therapeutic targets.

Benbow SJ et al. (2020) Hum Mol Genet "Synergistic toxicity between tau and amyloid drives neuronal dysfunction and neurodegeneration in transgenic C. elegans."

Kraemer BC, Benbow SJ, Strovas TJ, Darvas M, Saxton A

[Hum Mol Genet, 2020]

Aggregates of Aβ peptide and the microtubule-associated protein tau are key molecular hallmarks of Alzheimer's disease (AD). However, the interaction between these two pathologies and the mechanisms underlying disease progression have remained unclear. Numerous failed clinical trials suggest the necessity for greater mechanistic understanding in order to refine strategies for therapeutic discovery and development. To this end, we have generated a transgenic Caenorhabditis elegans model expressing both human Aβ1-42 peptide and human tau protein pan-neuronally. We observed exacerbated behavioral dysfunction and age-dependent neurodegenerative changes in the Aβ;tau transgenic animals. Further, these changes occurred in the Aβ;tau transgenic animals at greater levels than worms harboring either the Aβ1-42 or tau transgene alone and interestingly without changes to the levels of tau expression, phosphorylation or aggregation. Functional changes were partially rescued with the introduction of a genetic suppressor of tau pathology. Taken together, the data herein support a synergistic role for both Aβ and tau in driving neuronal dysfunction seen in AD. Additionally, we believe that the utilization of the genetically tractable C. elegans model will provide a key resource for dissecting mechanisms driving AD molecular pathology.

Wheeler, J.M. et al. (2019) International Worm Meeting "From worms to humans: using C. elegans as a model to discover mechanisms underlying neurodegenerative diseases and identify novel treatment targets."

Benbow, S.J., Kraemer, B.C., Wheeler, J.M., Waldherr, S.M., Kow, R.L.

[International Worm Meeting, 2019]

The protein tau forms toxic aggregates in diseases such as Alzheimer's and frontotemporal lobar degeneration (FTLD). Transgenic C. elegans expressing a mutant version of tau found in familial cases of FTLD have an Unc phenotype, and also exhibit neurodegeneration and accumulation of insoluble tau. We have conducted extensive forward genetic screens to identify both suppressors and enhancers of this Unc phenotype. Currently, we have identified mutations in over 20 genes that suppress tau phenotypes (sut genes), with a range of functions including RNA binding, cell cycle regulation, protein kinase/phosphatase, and tubulin genes. Our overall goal is to translate these findings into mammalian model systems, in order to identify novel targets for the treatment of tauopathy diseases. One example of this translational pipeline is our characterization of sut-2, encoding a zinc finger protein with well conserved homologs in all species from yeast (Nab2) to humans (ZC3H14, or MSUT2). sut-2 deletion restores locomotion back to wildtype levels, prevents neurodegeneration, eliminates insoluble tau, and extends lifespan in tau transgenic C. elegans. We generated knockout mice, and MSUT2 KO also suppresses tau phenotypes in a mammalian system. MSUT2 KO mice are resistant to the formation of tau pathology, and have improved cognitive abilities. By examining postmortem tissue from human AD patients, we also find that level of MSUT2 in the brain correlates with age of disease onset and severity of disease progression. We are currently developing inhibitors targeting MSUT2 as potential therapeutics for the treatment of tauopathy diseases. Our screen also identified a specific missense mutation in the tubulin gene mec-12 which suppresses tau phenotypes, although loss-of-function alleles of mec-12 do not show suppression. In order to translate this result into a mammalian system, we used CRISPR to generate a mouse line carrying the same missense mutation in the TUBA4A gene and are currently crossing this strain with tau transgenic mice. Finally, we identified xbp-1 as an enhancer of tau phenotypes; conversely, overexpression of xbp-1 in C. elegans acts as a suppressor. Preliminary data indicate that overexpression of XBP1 also reduces tau pathology in mice. In summary, we are using C. elegans as a model to explore the mechanisms of resilience against tau pathology, and translating these results into viable therapeutic targets for preclinical treatment trials.

Jin, Eugene et al. (2021) International Worm Meeting "Coordination of neuronal activity and transcriptional programs in motor circuit remodeling"

Jin, Yishi, Jin, Eugene

[International Worm Meeting, 2021]

Neuronal plasticity and circuit stability are fundamental properties of brain development and function. Activity-dependent changes to neuronal connectivity often occur within a defined time window, also known as a developmental plasticity window. How neuronal activity contributes to such precise timing of neural circuit rewiring is a central question in neuroscience. Ultrastructural connectomic studies that began nearly 50 years ago revealed that during the first larval stage, the C. elegans locomotor circuit undergoes dramatic synaptic rewiring known as 'DD synapse remodeling' as postembryonic motor neurons are born to establish the mature motor circuit (White et al., 1978). Live imaging studies subsequently showed that presynaptic terminals in DD motor neurons are progressively removed from the ventral side and new synapses are formed at dorsal locations from mid to late L1 stages (Hallam and Jin, 1998). The precise timing of DD synapse remodeling has been shown to depend on several transcriptional programs and can be modulated by neuronal activity. However, it remains unclear which form of neuronal activity affects the time window of this developmental plasticity, and how neuronal activity is molecularly coupled to transcription regulation. To address this, we are using fluorescently tagged reporters for in vivo detection of the key transcription factors, such as LIN-14 and UNC-30. To precisely determine L1 developmental stages, we use P cell nuclear migration and divisions using Nomarski optics (Sulston and Horvitz, 1977). We have also generated a nuclear calcium sensor to measure activity in DD neurons during synapse remodeling. We will present our detailed findings on the changes in nuclear calcium dynamics before, during and after DD synapse remodeling. References: Hallam, S.J., and Y. Jin. 1998. Nature. 395(6697):78-82. Sulston, J.E., and H.R. Horvitz. 1977. Dev. Biol. 56:110-156. White, J.G., D.G. Albertson, and M.A. Anness. 1978. Nature. 271:764-766.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Aoki, Scott Takeo et al. (2013) International Worm Meeting "Structural characterization of the P-granule protein scaffold."

Aoki, Scott Takeo, Kimble, Judith

[International Worm Meeting, 2013]

C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation. References: 1. Updike, D., Strome, S. (2010) J Androl 31: 53-60. 2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740. 3. Kawasaki, I., et al. (1998) Cell 94: 635-645. 4. Kawasaki, I., et al. (2004) Genetics 167: 645-661. 5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948. 6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..

Vidal , B. et al. (2019) International Worm Meeting "Decoding pharyngeal neuron fate specification."

Hobert, O., Gulez, B., Vidal , B., Reilly, M.

[International Worm Meeting, 2019]

The pharyngeal nervous system is composed of only 20 neurons belonging to 14 different types, which form a self-contained circuit that is almost independent from the somatic nervous system. This simplicity makes it possible to analyze it in a comprehensive way. Moreover, all pharyngeal neurons directly connect to end organs and can be considered polymodal with sensory-motor characteristics (see abstract by S.J. Cook and S.W.Emmons), a feature that is reminiscent of primitive nervous systems. Thus, understanding how pharyngeal neurons are specified during development might shed light on fundamental aspects of neuronal development. ceh-34, a homeodomain transcription factor of the Six family, is continuously expressed in all pharyngeal neurons and no other neurons outside of the pharynx. Remarkably, we have found that in ceh-34 mutants, pharyngeal neurons are generated, but fail to express a wide array of terminal identity genes, including neurotransmitter pathway genes, indicating that ceh-34 acts as a pharyngeal neuron master regulator ("terminal selector"). Moreover, a conditional AID-based allele demonstrates that ceh-34 is continuously required during the life of the worm to maintain pharyngeal neuron identity. We hypothesize that ceh-34 acts together with other transcription factors to form a combinatorial code that gives each pharyngeal neuron its unique identity. We have found several other homeodomain transcription factors expressed in subsets of pharyngeal neurons (see abstract by M. Reilly and O.Hobert) and we are doing a mutant analysis to test whether they also play a role in the pharyngeal nervous system specification. Moreover, we are performing forward genetic screens to find additional factors in an unbiased way. So far we have identified four mutants that appear to affect I2 neuron identity. We are in the process of pinpointing the causal molecular lesions and further characterizing these new mutants. We hope our efforts will lead to a comprehensive understanding of the regulatory code that dictates pharyngeal neuron development.

Watanabe, Shigeki et al. (2011) International Worm Meeting "Two endocytic pathways revealed by capturing channelrhodopsin induced synaptic transmission."

Jorgensen, Erik, Hollopeter, Gunther, Gu, Mingyu, Davis, Wayne, Watanabe, Shigeki, Liu, Qiang

[International Worm Meeting, 2011]

To sustain release of neurotransmitters at high rates of stimulation, synaptic vesicles need to be recycled locally at a synapse. Two major pathways for endocytosis were identified nearly 40 years ago: clathrin-scaffolds[1] and kiss-and-run[2]. Clathrin-mediated endocytosis is a slow process, which seems to take place distant from release sites. On the other hand, kiss-and-run occurs at release sites immediately after neurotransmission. The existence of either pathway at synapses has not been satisfactorily demonstrated[3]. Moreover, molecular mechanisms behind these pathways are poorly understood[3]. To address these issues, we need to observe synaptic vesicles as well as their associated proteins in action at nanoscale resolution. We developed two techniques that allow such an observation. First, we combined optogenetics with electron microscopy to induce synaptic transmission and capture rapid events at nanoscale resolution. Specifically, we expressed a variant of channelrhodopsin, ChIEF, in the C. elegans acetylcholine neurons, and stimulated them at various times before the high-pressure freezing. Using this technique, we identified two endocytic pathways that act at different times and in different parts of the synapse: a slow process distant from the dense projection and a fast process near the dense projection. Second, we developed a correlative super-resolution fluorescence microscopy and electron microscopy (fEM) imaging technique[4] to identify molecules that act in the processes. We found that the adaptor protein AP2 is localized to both identified endocytic sites, suggesting that AP2 may be functioning in both processes. By combining these two techniques, we are beginning to understand how endocytosis takes place at synapses. References: [1]Heuser, J.E. & Reese, T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol 57, 315-344 (1973). [2]Ceccarelli, B., Hurlbut, W.P. & Mauro, A. Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J. Cell Biol 54, 30-38 (1972). [3]Royle, S.J. & Lagnado, L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules. Traffic 11, 1489-1497 (2010). [4]Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80-84 (2011).

Edgley ML et al. (1991) International C. elegans Meeting "Caenorhabditis genetics center."

Riddle DL, Edgley ML, Estevez AOZ

[International C. elegans Meeting, 1991]

The CGC has been in operation for 12 years, supplying Caenorhabditis strains and information to researchers. It is responsible for coordination of C. elegans genetic nomenclature, and acts as the central clearing house for naming strains, genes and alleles. Information distributed includes the genetic map, genetic map data, strain information, a bibliography of 1300 research articles and book chapters, and the C. elegans research newsletter, the Worm Breeder's Gazette, which is distributed to 450 subscribers worldwide. A collection of 1550 strains has been established, representing 30 wildtype isolates of C. elegans, wild-type strains of C. briggsae, C. remanei and C. vulgariensis, as well as alleles of 773 mutant genes and 254 chromosome rearrangements. Stocks are stored permanently in liquid nitrogen. Approximately 1350 different strains have been distributed to research laboratories in 7800 separate mailings. Investigators in 210 laboratories in 21 countries have either received strains from the CGC or contributed strains to the CGC collection. The current version of the C. elegans genetic map includes 26 pages, and contains 920 genes and 320 rearrangements. It is produced using the computer drawing program Designer (Micrografx, Inc., Richardson TX), which runs under Microsoft Windows on IBM-compatible microcomputers. The map is published biannually in Genetic Maps (S.J. O'Brien, editor; Cold Spring Harbor Laboratory) and semi-annually in the Worm Breeder's Gazette. Caenorhabditis strains and information are available upon request. All strain, bibliographic and genetic information is maintained on a microcomputer database system, and the various files are available as printouts or data on diskette. Operation of the CGC will be transferred to other investigators at the end of the current CGC contract (September 29,1992). One possible plan emerged at the last C. elegans meeting, in which Bob Herman will assume responsibility for the strain collection, and Jonathan Hodgkin will assume responsibility for the genetic map. Plans for continuation of other CGC services remain to be made. New developments in the use of computers to integrate all information on the worm will enhance the usefulness of data collected by the CGC (see Thierry-Mieg, Durbin, Schatz and Ward, this meeting). The CGC is supported by the NIH National Center for Research Resources.