Biochemical and cellular analysis of human variants of the DYT1 dystonia protein, TorsinA/TOR1A.

Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA(+) protein family that localizes to the endoplasmic reticulum lumen and nuclear envelope. This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients: R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia.

Generation of a novel rodent model for DYT1 dystonia.

A mutation in the coding region of the Tor1A gene, resulting in a deletion of a glutamic acid residue in the torsinA protein (∆ETorA), is the major cause of the inherited autosomal-dominant early onset torsion dystonia (DYT1). The pathophysiological consequences of this amino acid loss are still not understood. Currently available animal models for DYT1 dystonia provided important insights into the disease; however, they differ with respect to key features of torsinA associated pathology. We developed transgenic rat models harboring the full length human mutant and wildtype Tor1A gene. A complex phenotyping approach including classical behavioral tests, electrophysiology and neuropathology revealed a progressive neurological phenotype in ∆ETorA expressing rats. Furthermore, we were able to replicate key pathological features of torsinA associated pathology in a second species, such as nuclear envelope pathology, behavioral abnormalities and plasticity changes. We therefore suggest that this rat model represents an appropriate new model suitable to further investigate the pathophysiology of ∆ETorA and to test for therapeutic approaches.

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia.

BACKGROUND: Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. NEW METHOD: The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. RESULTS: We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. COMPARISON WITH EXISTING METHOD: The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. CONCLUSION: We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment.