Human Molecular Genetics Advance Access published online on February 7, 2008
Human Molecular Genetics, doi:10.1093/hmg/ddn032
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siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells
1 Molecular Neurogenetics Unit, Department of Neurology, and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA 2 Alnylam Pharmaceuticals, Cambridge, MA 02142, USA
* Correspondence: Xandra O. Breakefield, PhD., Molecular Neurogenetics Unit, Massachusetts General Hospital-East, 13th Street, Building 149, Charlestown, MA, 02129, USA, Phone 617-726-5728, Fax 617-724-1537, E-mail: breakefield{at}hms.harvard.edu
Received December 6, 2007; Revised January 29, 2008; Accepted January 29, 2008
Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the carboxy terminal region (torsinA
E). TorsinA is an AAA + protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE) apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinAE as compared to control fibroblasts expressing only torsinA. In this study small interfering RNA oligonucleotides (siRNA) were identified which downregulate levels of torsinA or torsinAE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared to the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinAE message in DYT fibroblasts increased luciferase secretion as compared to cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinAE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinAE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.
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