Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.
Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington's disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.
Progressive neurodegeneration in HD involves pathogenic mutant huntingtin with an abnormal stretch of polyQ encoded by CAG trinucleotide repeats1,2,3. Mutant huntingtin proteins with more than 37 glutamine repeats are prone to aggregate and accumulate in the brains of HD patients and animal models4,5, which ultimately leads to neurodegeneration6. Despite the lack of clarity on the roles of polyQ aggregates in disease pathology5, the inhibition of polyQ aggregation and its associated toxicity is a useful therapeutic strategy for HD and other polyQ diseases4,7,8.
Due to the conservation in neuronal signaling pathways and easy-to-construct transgenic disease models, Caenorhabditis elegans has been widely used as a major model organism for the investigation of neurological disorders9,10,11,12. For example, transgenic C. elegans models expressing aggregation-prone polyQ expansions can objectively mimic HD-like features such as selective neuronal cell loss, cytoplasmic aggregate formation, and behavioral defects13. Investigation of the potential effects of test samples to reverse these phenotypes in established polyQ nematode models has led to the identification of a variety of promising therapeutic candidates, e.g., polysaccharides7,14,15, oligosaccharides16, natural small molecules17,18, and herbal extracts and formulas19,20.
Described here are two main polyQ C. elegans models and relevant protocols for potential applications as exemplified by the study on astragalan, a polysaccharide isolated from Astragalus membranaceus7. For the polyQ aggregation assay in C. elegans, the model used is the transgenic strain AM141, which shows fluorescent puncta dispersed in its body wall muscle when reaching adulthood due to the expression of the Q40::YFP fusion protein, a polyQ tract of 40 residues (polyQ40) fused to yellow fluorescent protein (YFP)21,22. The strain HA759 was used to examine neuronal survival and chemoavoidance behavior as it expresses both green fluorescent protein (GFP) and Htn-Q150 (a human huntingtin-derived polyQ tract of 150 residues) strongly in ASH neurons but weakly in other neurons, resulting in progressive neurodegeneration and ASH cell death7,13. A comprehensive summary of the neuroprotective potential of therapeutic candidates is provided by integrating results from different assays.
As polyQ aggregation and proteotoxicity are important features of polyQ disorders, such as Huntington’s disease13, we recommend the use of multiple models and methods to comprehensively evaluate the neuroprotective capacity of test compounds, including the polyQ aggregation assay in the AM141 strain, the ASH neuronal survival assay in the HA759 strain, and the chemosensory avoidance assay in the HA759 strain. The protocols presented here have been used to evaluate the neuroprotective capacities of test samples against polyQ toxicity, including inhibitory effects on both polyQ aggregation and associated neurotoxicity7,14,15,16,17,19,20, demonstrating their potential in drug discovery for HD and other polyQ diseases.
An automated imaging and analysis system is introduced for the detection and counting of polyQ aggregates in the polyQ aggregation assay. This method has the advantages of being high-throughput and time-efficient and results in significantly reduced subjective errors in the laborious counting process. For an entire 384-well plate, it only takes <1 h to finish image acquisition and analysis. However, the conventional microscopic imaging method has also shown similar performance in this laboratory without using the automated imaging device7.
A total of 100-150 nematodes per treatment are recommended in a typical Q40::YFP aggregation assay for each time point, which can be performed in replicate wells containing 10-15 nematodes each. However, it should be noted that L1 larvae may be more sensitive to some treatments or higher concentrations. Therefore, higher doses of test compounds might inhibit their growth, leading to false-positive results due to slow growth and, thus, delayed polyQ aggregation. Usually, a food clearance assay can be performed to address this concern and ensure the appropriate concentration range of test compounds23.
The HA759 transgenic nematodes used in polyQ neurotoxicity assays coexpress OSM-10::GFP and Htn-Q150, making it possible to unambiguously identify bilateral ASH sensory neurons. Hence, ASH neuron survival is evaluated by the presence or absence of GFP expression; usually, ~40-75% of ASH neurons in the control nematodes are dead23,24. Interestingly, the pqe-1 (polyglutamine enhancer-1) genetic mutant background in the HA759 strain (pqe-1;Htn-Q150) accelerates polyQ-mediated toxicity, leading to the death of most ASH neurons within three days, even at 15 °C, and therefore this strain is grown at 15 °C for the neuronal survival assay, as previously reported23,24.
Functional loss of ASH neurons in HA759 nematodes may occur before the detection of cell death and protein aggregates13; therefore, the osmotic avoidance behavior assay is essential for the assessment of polyQ-mediated toxicity. To minimize the potential impact of less active HA759 nematodes at low temperature on behavioral experiments, the avoidance assay plates are incubated in a humidified 23 °C incubator rather than at 15 °C as in the neuronal survival assay using this strain. In addition, it has been reported that Htn-Q150/OSM-10::GFP transgenic nematodes are highly sensitive to nose touch; hence, an alternative detection of ASH neuron function is the nose touch assay13.
The authors have nothing to disclose.
We thank former members of the Huang Lab who have helped develop and improve the protocols used in this paper, particularly, Hanrui Zhang, Lingyun Xiao, and Yanxia Xiang. This work was supported by the 111 Project (grant number B17018) and the Natural Science Foundation of Hebei Province (grant number H2020207002).
C. elegans strains | |||
AM141 rmIs133 [unc-54p::Q40::YFP] |
Caenorhabditis Genetics Center (CGC) | https://cgc.umn.edu/strain/AM141 | |
HA759 rtIs11 [osm-10p::GFP + osm-10p::HtnQ150 + dpy-20(+)] |
Caenorhabditis Genetics Center (CGC) | https://cgc.umn.edu/strain/HA759 | |
E. coli strains | |||
NA22 | Caenorhabditis Genetics Center (CGC) | https://cgc.umn.edu/strain/NA22 | |
OP50 | Caenorhabditis Genetics Center (CGC) | https://cgc.umn.edu/strain/OP50 | |
Reagent | |||
Agar | Shanghai EKEAR Bio-Technology Co., Ltd. | EQ1001-500G | https://www.ekear.com |
Agarose | Biowest | 111860 | |
Butanedione | Sinopharm Chemical Reagent Co., Ltd. | 80042427 | https://www.reagent.com.cn/goodsDetail/d027c00e64c9404d9aa41391fbb59 5d0 |
Cholesterol | Sigma-Aldrich | C8667 | https://www.sigmaaldrich.cn/CN/zh/product/sigma/c8667?context=product |
Glycerol | Aladdin Co., Ltd. | G116203 | https://www.aladdin-e.com/zh_cn/g116203.html |
Peptone | Guangdong HuanKai Microbial Science and Technology Co., Ltd. | 050170B | https://www.huankai.com/show/21074.html |
Sodium azide | Sinopharm Chemical Reagent Co., Ltd. | 80115560 | https://www.reagent.com.cn/goodsDetail/5e981aa807664e26af 551e96ff5f07cd |
Sodium hydroxide | Sinopharm Chemical Reagent Co., Ltd. | 10019718 | https://www.reagent.com.cn/goodsDetail/450dfdb1132a4d8a817 d3d8c68ec25e6 |
Sodium hypochlorite solution | Guangzhou Chemical Reagent Factory | 7681-52-9 | http://www.chemicalreagent.com/product/DetailProduct.aspx?id=125 |
Tryptone | Oxoid Ltd. | LP0042B | https://www.thermofisher.cn/order/catalog/product/LP0042B#/LP0042B |
Yeast extract | Oxoid Ltd. | LP0021B | https://www.thermofisher.cn/order/catalog/product/LP0021B#/LP0021B |
Equipment | |||
384-well cell culture plate | Nest Biotechnology Co., Ltd. | 761001 | https://www.cell-nest.com/page94?_l=en&product_id=85 |
48-well cell culture plate | Nest Biotechnology Co., Ltd. | 748001 | https://www.cell-nest.com/page94?_l=en&product_id=85 |
90 mm Petri dish | Sangon Biotech (Shanghai) Co., Ltd. | F611003 | https://www.sangon.com/productDetail?productInfo.code=F611003 |
Autoclave | Panasonic | MLS-3781L-PC | |
Dissecting microscope | ChongQing Optical Co., Ltd. | ZSA0745 | http://www.coicuop.com/plus/view.php?aid=64 |
Fluorescence microscope | Guangzhou Micro-shot Optical Technology Co., Ltd. | Mshot MF31-LED | https://www.mshot.com/article/442.html |
High-content imaging system | Molecular Devices | ImageXpress Pico | https://www.moleculardevices.com/products/cellular-imaging-systems#High-Content-Imaging |
Microcentrifuge | GeneCompany | GENESPEED X1 | https://www.genecompany.com/index.php/Home/Goods/goodsdetails/gid/189.html |
Microscope digital camera | Guangzhou Micro-shot Optical Technology Co., Ltd. | MS60 | https://www.mshot.com/article/677.html |
Microwave | Midea Corp. | M1-211A | https://www.midea.cn/10000/10000000001 00511264425.html |
Parafilm M | Sigma-Aldrich | P7793-1EA | https://www.sigmaaldrich.cn/CN/en/product/sigma/p7793?context=product |
Shaker | Zhicheng Inc. | ZWY-2102C | http://www.zhicheng.net/Product/0865291356.html |
Software | |||
Image acquisition and analysis software | Molecular Devices | MetaXpress | https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metaxpress |
OriginPro | OriginLab Corp. | Version 9.8.5.204 | 1. Software introduction: https://www.originlab.com/index.aspx?go=Products/Origin 2. Instruction for creating a radar chart: https://www.originlab.com/doc/Origin-Help/RadarChart-Graph 3. Video tutorial for creating a radar chart: https://www.originlab.com/videos/details.aspx?pid=1813 |