सी एलिगेंस Dopamine न्यूरॉन अध: पतन परख की संभावित पार्किंसंस रोग जीन की मान्यता के लिए आवेदन
Summary
इस वीडियो यह दर्शाता है कि कैसे सी एलिगेंस का उपयोग करने के लिए पार्किंसंस रोग के लिए एक मॉडल के रूप में डोपामिनर्जिक न्यूरॉन neurodegeneration आकलन. इसके अलावा, आनुवंशिक स्क्रीन कारक है कि या तो अध: पतन को बढ़ाने या neuroprotective हैं की पहचान करने के लिए उपयोग किया जाता है.
Abstract
Improvements to the diagnosis and treatment of Parkinson’s disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.
Protocol
Discussion
dopamine न्यूरॉन्स की उम्र पर निर्भर हानि पार्किंसंस रोग के नैदानिक पहचान है और है या एक प्रोटीन अल्फा synuclein कहा जाता है के संचय misfolding के साथ संबद्ध किया गया है है. यहाँ हम प्रदर्शन कैसे लेबल करने के लिए, एक फ्लोरोसेंट …
Acknowledgements
हम सभी काल्डवेल लैब के सदस्यों के सहकारी भावना को स्वीकार चाहते हैं. आंदोलन विकारों अनुसंधान प्रयोगशाला में dystonia Bachmann – स्ट्रॉस और पार्किंसंस फाउंडेशन, संयुक्त पार्किंसंस फाउंडेशन, अमेरिकी पार्किंसंस रोग एसोसिएशन, अलबामा के पार्किंसंस रोग एसोसिएशन, माइकल जे फॉक्स फाउंडेशन पार्किंसंस अनुसंधान के लिए, और एक अंडर ग्रेजुएट रिसर्च द्वारा समर्थित किया गया
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Agarose | Ultrapure | Invitrogen | 15510-027 | |
| Coverglass 20×30 mm | Fisher | 12-548-5A | ||
| Microscope Slides | Plain, 3×1″ | Fisher | 12-549 | |
| Dissecting with Fluorescence | Microscope | Nikon | SMZ800 | |
| Dissecting | Microscope | Nikon | SMZ645 | |
| Epifluorescent | Microscope | Nikon | Model E-800 | |
| Filter Cube, GFP HYQ | Endow Bandpass | Chroma Technology |
References
- Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans. J Neurosci. 25, 3801-3812 (2005).
- Hamamichi, S., Rivas, R. N., Knight, A. L., Cao, S., Caldwell, K. A., Caldwell, G. A. Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson’s disease model. PNAS. 105, 728-733 (2008).
- Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M., Lindquist, S. alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science. 313, 324-328 (2006).
- Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. -. C., McCaffery, J. M., Barlowe, C., Lindquist, S. The Parkinson’s disease protein alpha-synuclein disrupts cellular Rab homeostasis. PNAS. 105, 145-150 (2008).
- Caldwell, G. Integrated Genomics: A Discovery-Based Laboratory Course. , (2006).
- Brenner, S. The genetics of Caenorhabditis elegans. Genetics. 77, 71-94 (1974).
- Gandhi, S., Santelli, J., Mitchell, J. D. H., Stiles, J. W., Sanadi, D. R. A simple method for maintaining large, aging populations of Caenorhabditis elegans. Mechanisms of Ageing and Development. 12, 137-150 (1980).
