Early onset torsion dystonia (DYT1) is a dominantly inherited movement disorder characterized by sustained, involuntary muscle contractions and abnormal posturing (1
). Most cases are caused by a specific deletional mutation (ΔGAG) in the DYT1
) gene encoding torsinA, which results in loss of a glutamic acid residue in the C-terminal region (2
). TorsinA is a member of the superfamily of ATPases associated with a variety of activities (AAA+), a group of chaperone proteins involved in processing, degradation, movement and dynamic associations of other proteins (3
). AAA+ proteins typically form homomeric six member ring structures which bind substrate in the ATP bound state. The normal function of torsinA has not yet been delineated and different studies implicate various potential functions in mammalian cells, including the architecture of the nuclear envelope (NE) (5
), neurite extension (7
), cell adhesion (7
), processing of proteins through the secretory pathway (9
) and protection of cells from toxic insults and abnormal proteins (12
). Many of these functions may involve protein interactions in the endoplasmic reticulum (ER). Further, a number of studies indicate that torsinAΔE may act by inhibiting the normal function of torsinA (reviewed in 18
). The presence of a specific mutation, a GAG deletion in the coding region of the DYT1 gene encoding torsinA (2
), provides the opportunity for development of rational therapy for this disease using allele-specific silencing of the torsinAΔE mRNA.
In the present study, torsinA function was monitored by assessing the status of the secretory pathway. Several studies support a function for torsinA in processing of proteins through the secretory pathway. Overexpression of torsinA in cultured cells inhibited processing of polytopic plasma membrane proteins, such as the dopamine transporter (DAT) (9
), and overexpression of a torsinA homolog in nematodes decreased expression of a DAT–GFP fusion construct (17
). Overexpression of torsinAΔE also caused entrapment of the vesicular monoamine transporter in membranous inclusions (10
) formed by overexpression of torsinAΔE (19
). In recent studies, we have shown that fibroblasts from DYT1 patients infected with a lentivirus vector encoding the secretory reporter protein, Gluc had a reduced rate of secretion of luciferase into the medium when compared with control human fibroblasts (11
RNA interference (RNAi) has proven to be an effective means to silence gene expression by catalyzing the degradation of mRNAs (21
). RNAi can be mediated by chemically modified small interfering RNA (siRNA) oligonucleotides (typically about 22 bp) or by short hairpin (sh) RNAs delivered via viral vectors. Gonzalez-Alegre et al
) initially demonstrated allele-specific silencing of the torsinAΔE mutant (mt) and torsinA wild-type (wt) mRNAs in cultured cells by co-transfection of specific siRNAs using expression cassettes for torsinA-GFP and torsinAΔE-GFP fusion proteins. In subsequent studies, this group demonstrated allele-specific silencing using a lentivirus vector expressing shRNAs, including suppression of endogenous torsinA in mammalian neurons (25
). Kock et al
) demonstrated that selective shRNA silencing of torsinAΔE expression using a lentivirus vector could block the formation of inclusion bodies generated by overexpression of this mt protein in primary neurons.
In the present study, we evaluated the ability of siRNA to selectively downregulate endogenous levels of torsinA and torsinAΔE in cultured fibroblasts from DYT1 patients and controls and the consequent effects on secretion of Gluc from cells. Inhibition of torsinA expression in control cells led to a decrease in Gluc secretion, whereas inhibition of torsinAΔE or overexpression of torsinA normalized secretion in patient cells.