Zhang S et al. (2013) WormBook "Cell isolation and culture."

Zhang S, Kuhn JR

[WormBook, 2013]

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices.

Zhang S et al. (2011) PLoS One "Isolation and culture of larval cells from C. elegans."

Kuhn JR, Zhang S, Banerjee D

[PLoS One, 2011]

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8x10(4) cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

Zhang S et al. (2004) Cell "Combinatorial marking of cells and organelles with reconstituted fluorescent proteins."

Ma C, Chalfie M, Zhang S

[Cell, 2004]

Expression of GFP and other fluorescent proteins depends on cis-regulatory elements. Because these elements rarely direct expression to specific cell types, GFP production cannot always be sufficiently limited. Here we show that reconstitution of GFP, YFP, and CFP previously split into two polypeptides yields fluorescent products when coexpressed in C. elegans. Because this reconstitution involves two components, it can confirm cellular coexpression and identify cells expressing a previously uncharacterized promoter. By choosing promoters whose expression patterns overlap for a single cell type, we can produce animals with fluorescence only in those cells. Furthermore, when one partial GFP polypeptide is fused with a subcellularly localized protein or peptide, this restricted expression leads to the fluorescent marking of cellular components in a subset of cells.

Zhang S et al. (2021) Environ Pollut "Responses of Caenorhabditis elegans to various surface modifications of alumina nanoparticles"

Zhang Z, Xu Y, Mao X, Chu Q, Zhang S, Zhang M

[Environ Pollut, 2021]

The surface modifications of nanoparticles (NPs), are well-recognized parameters that affect the toxicity, while there has no study on toxicity of Al(2)O(3) NPs with different surface modification. Therefore, for the first time, this study pays attention to evaluating the toxicity and potential mechanism of pristine Al(2)O(3) NPs (p-Al(2)O(3)), hydrophilic (w-Al(2)O(3)) and lipophilic (o-Al(2)O(3)) modifications of Al(2)O(3) NPs both in vitro and in vivo. Applied concentrations of 10, 20, 40, 80,100 and 200 μg/mL for 24 h exposure on Caenorhabditis elegans (C. elegans), while 100 μg/mL of Al(2)O(3) NPs significantly decreased the survival rate. Using multiple toxicological endpoints, we found that o-Al(2)O(3) NPs (100 μg/mL) could induce more severe toxicity than p-Al(2)O(3) and w-Al(2)O(3) NPs. After uptake by C. elegans, o-Al(2)O(3) NPs increased the intestinal permeability, easily swallow and further destroy the intestinal membrane cells. Besides, cytotoxicity evaluation revealed that o-Al(2)O(3) NPs (100 μg/mL) are more toxic than p-Al(2)O(3) and w-Al(2)O(3). Once inside the cell, o-Al(2)O(3) NPs could attack mitochondria and induce the over-production of reactive oxygen species (ROS), which destroy the intracellular redox balance and lead to apoptosis. Furthermore, the transcriptome sequencing and RT-qPCR data also demonstrated that the toxicity of o-Al(2)O(3) NPs is highly related to the damage of cell membrane and the imbalance of intracellular redox. Generally, our study has offered a comprehensive sight to the adverse effects of different surface modifications of Al(2)O(3) NPs on environmental organisms and the possible underlying mechanisms.

Zhang S et al. (2002) Cell "Drosophila atrophin homolog functions as a transcriptional corepressor in multiple developmental processes."

Xu T, Lee J, Zhang S, Xu L

[Cell, 2002]

Dentatorubral-pallidoluysian atrophy is a progressive neurodegenerative disease caused by the expansion of a polyglutamine repeats within the Atrophin-1 protein. The in vivo function of Atrophin-1 is unknown. We have characterized a Drosophila gene encoding an Atrophin family protein. Analysis of mutant phenotypes indicates that Drosophila Atrophin is required in diverse developmental processes including early embryonic patterning. Drosophila Atrophin genetically interacts with the transcription repressor even-skipped and is required for its repressive function in vivo. Drosophila Atrophin directly binds to Even-skipped in vitro. Furthermore, both human Atrophin-1 and Drosophila Atrophin repress transcription in vivo when tethered to DNA, and poly-Q expansion in Atrophin-1 reduces this repressive activity. We propose that Atrophin proteins function as versatile transcriptional corepressors and discuss a model that deregulation of transcription may contribute to the pathogenesis of neurodegeneration.

Zhang S et al. (2004) Development "Caenorhabditis elegans TRPV ion channel regulates 5HT biosynthesis in chemosensory neurons."

Zhang S, Sze JY, Blanco G, Sokolchik I

[Development, 2004]

Serotonin (5HT) is a pivotal signaling molecule that modulates behavioral and endocrine responses to diverse chemical and physical stimuli. We report cell-specific regulation of 5HT biosynthesis by transient receptor potential V (TRPV) ion channels in C elegans. Mutations in the TRPV genes osm-9 or ocr-2 dramatically downregulate the expression of the gene encoding the 5HT synthesis enzyme tryptophan hydroxylase (tph-1) in the serotonergic chemosensory neurons ADF, but neither the mutation nor the double mutation of both channel genes affects other types of serotonergic neurons. The TRPV genes are expressed in the ADF neurons but not in other serotonergic neurons, and act cell-autonomously to regulate a neuron-specific transcription program. Whereas in olfactory neurons OSM-9 and OCR-2 function is dependent on ODR-3 Galpha, the activity of ODR-3 or two other Ga proteins expressed in the ADF neurons is not required for upregulating tph-1 expression, thus the TRPV ion channels in different neurons may be regulated by different mechanisms. A gain-of-function mutation in CaMKII UNC-43 partially suppresses the downregulation of tph-1 in the TRPV mutants, thus CaMKII may be an effector of the TRPV signaling. Mutations in the TP.PV genes cause worms developmentally arrest at the Dauer stage. This developmental defect is due in part to reduced 5HT inputs into daf-2/insulin

Zhang S et al. (2020) Front Endocrinol (Lausanne) "Caenorhabditis elegans as a Useful Model for Studying Aging Mutations."

Zhang S, Li Z, Zhou T, Wang G, Li F

[Front Endocrinol (Lausanne), 2020]

The <i>Caenorhabditis elegans</i> genome possesses homologs of about two-thirds of all human disease genes. Based on its physiological aging characteristics and superiority, the use of <i>C. elegans</i> as a model system for studies on aging, age-related diseases, mechanisms of longevity, and drug screening has been widely acknowledged in recent decades. Lifespan increasing mutations in <i>C. elegans</i> were found to delay aging by impinging several signaling pathways and related epigenetic modifications, including the insulin/IGF-1 signaling (IIS), AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR) pathways. Interestingly, dietary restriction (DR) has been shown to increase the lifespan of numerous metazoans and protect them from multiple age-related pathologies. However, the underlying molecular mechanisms are unclear. In recent decades, <i>C. elegans</i> has been used as a unique model system for high-throughput drug screening. Here, we review <i>C. elegans</i> mutants exhibiting increased in lifespan and age-dependent changes under DR, as well as the utility of <i>C. elegans</i> for drug screening. Thus, we provide evidence for the use of this model organism in research on the prevention of aging.

Zhang S et al. (2015) Iran J Public Health "The Analysis of Gene Expression on Fertility Decline in Caenorhabditis elegans after the Treatment with 5-Fluorouracil."

Zhang Y, Bao G, Fu X, Wu J, Wang X, Lu Z, Wang M, Liu X, Jing T, Liang H, Zhang S, Chen G

[Iran J Public Health, 2015]

BACKGROUND: 5-Fluorouracil could lead to a decline in fertility in Caenorhabditis elegans. The aim of this study was to describe the mechanisms underlying such an altered fertility phenotype and to illustrate the specific genes and pathways that are involved in the related phenotypic changes in C. elegans. METHODS: We isolated total RNA from the samples and used a new method called Digital Gene Expression (DGE), which can rapidly identify genes with altered transcript levels. The random genes were confirmed by real-time RT-PCR. RESULTS: We analyzed the results of two methods to draw conclusions based on a comparison between C. elegans and other harmful parasites. Compared with controls, 1147 genes were up-regulated, and 1067 were down-regulated. Overall, 101 up-regulated genes had a log2 ratio higher than 8, whereas the log2 ratio of 141 down-regulated genes was higher than 8. After mapping to the reference database, 4 pathways were confirmed to be involved in this phenomenon, with statistically significant participation from 19 genes. CONCLUSION: For the first time, the transcript sequence of 5-Fu-treated worms and controls was detected. We found that 4 possible pathways, i.e., ECM-receptor interaction pathway, TGF-beta signaling pathway, Focal adhesion and Hypertrophic cardiomyopathy, may be involved in the number decline in the embryos of C. elegans. Specifically, the ECM-receptor interaction pathway and Focal adhesion may be very important pathways that alter the reproduction of C. elegans.

Zhang, S. et al. (2013) International Worm Meeting "Neutral cholesterol ester hydrolase 1 is protective against a-synuclein-induced toxicity in C. elegans."

Caldwell, G. A., Zhang, S., Caldwell, K. A.

[International Worm Meeting, 2013]

Aging is the most identified risk factor for Parkinson's disease (PD). In C. elegans, we model a cellular aspect of PD by overexpressing a-synuclein (a-syn), a protein that forms inclusions in the brains of PD patients. Nematodes overexpressing a-syn::GFP in the body wall muscles exhibit age-dependent protein aggregates. We can greatly reduce these a-syn-induced inclusions by introducing a mutation in the worm insulin-like signaling receptor gene, daf-2. To identify components in the daf-2 pathway relevant to a-syn accumulation, we performed an RNAi screen for genes that, when knocked down, resulted in enhanced a-syn misfolding in daf-2 mutants. In total, 60 candidates were identified; one of these was an open reading frame Y43F8A.3. We were curious to know if the corresponding gene product would also modulate a-syn toxicity in a neuronal model of PD. C. elegans expressing a-syn in the dopaminergic (DA) neurons of the nematode exhibit age- and dose-dependent neurodegeneration. DA expression of Y43F8A.3 rescues a-syn-induced neurodegeneration. Y43F8A.3 is predicted to encode an ortholog of human neutral cholesterol ester hydrolase 1 (NCEH1). In general, cholesterol ester hydrolase can convert esterified cholesterol to free cholesterol and fatty acids. Considering that neuroprotection by Y43F8A.3 may be associated with a change of cholesterol levels, we further hypothesize that genetic manipulation of a cholesterol regulatory transcriptional factor will result in similar effect on a-syn inclusions. SBP-1 is the worm homolog of human SREBP1 (Sterol Regulatory Element Binding Protein 1). SREBP1 is the transcription factor regulating targets involved in cholesterol homeostasis, such as lipid synthesis and cellular trafficking genes. When knocked down, sbp-1 also enhanced a-syn toxicity in the DA neuron PD model. Additionally, varying concentrations of cholesterol in the medium have different effects on a-syn toxicity on DA neurons. Lastly, we find that knockdown of genes involved in cholesterol trafficking also enhanced a-syn toxicity on DA neurons. Collectively, cholesterol levels may have an impact on a-syn toxicity in C. elegans DA neurons, thus representing a putative metabolic effector of neurodegeneration.

Zhang S et al. (2017) Hum Mol Genet "NCEH-1 modulates cholesterol metabolism and protects against -synuclein toxicity in a C. elegans model of Parkinson's disease."

Caldwell KA, Glukhova SA, Caldwell GA, Zhang S

[Hum Mol Genet, 2017]

Parkinson's disease (PD) is an aging-associated neurodegenerative disease affecting millions worldwide. Misfolding, oligomerization and accumulation of the human -synuclein protein is a key pathological hallmark of PD and is associated with the progressive loss of dopaminergic neurons over the course of aging. Lifespan extension via the suppression of IGF-1/insulin-like signaling (IIS) offers a possibility to retard disease onset through induction of metabolic changes that provide neuroprotection. The nceh-1 gene of Caenorhabditis elegans encodes an ortholog of neutral cholesterol ester hydrolase 1 (NCEH-1), an IIS downstream protein that was identified in a screen as a modulator of -synuclein accumulation in vivo. The mechanism whereby cholesterol metabolism functionally impacts neurodegeneration induced by -synuclein is undefined. Here we report that NCEH-1 protects dopaminergic neurons from -synuclein-dependent neurotoxicity in C. elegans via a mechanism that is independent of lifespan extension. We discovered that the presence of cholesterol, LDLR-mediated cholesterol endocytosis, and cholesterol efflux are all essential to NCEH-1-mediated neuroprotection. In protecting from -synuclein neurotoxicity, NCEH-1 also stimulates cholesterol-derived neurosteroid formation and lowers cellular reactive oxygen species in mitochondria. Collectively, this study augments our understanding of how cholesterol metabolism can modulate a neuroprotective mechanism that attenuates -synuclein neurotoxicity, thereby pointing toward regulation of neuronal cholesterol turnover as a potential therapeutic avenue for PD.