Mutant torsinA, which causes early‐onset primary torsion dystonia, is redistributed to membranous structures enriched in vesicular monoamine transporter in cultured human SH‐SY5Y cells

Abstract

A single GAG deletion in the DYT1 gene causes primary early-onset, generalized torsion dystonia. The DYT1 protein product, torsinA, belongs to the AAA+ family of proteins. When overexpressed, wild-type torsinA localizes mainly to the endoplasmic reticulum, whereas the mutant forms inclusions of unclear biogenetic origin. In this study, overexpressed wild-type torsinA in human neuroblastoma (SH-SY5Y) cell lines was distributed throughout the cell body and colocalized with a marker for the endoplasmic reticulum, confirming it is an endoplasmic reticulum protein. However, mutant torsinA showed perinuclear staining and formed distinct globular inclusions, which did not colocalize with endoplasmic reticulum markers. Immunoelectron microscopy of the mutant torsinA inclusions revealed membrane whorls staining for torsinA, as well as labeling of lamellae, isolated bilayers, and perinuclear membranes. This finding shows that mutant torsinA redistributes to specific membranous structures, which may represent different stages of maturation of the intracellular inclusions. The mutant torsinA-containing bodies were immunoreactive for vesicular monoamine transporter 2 (VMAT2). VMAT2 expression is important for the exocytosis of bioactive monoamines in neurons. Abnormal processing, transport, or entrapment of VMAT2 within the mutant torsinA membranous inclusions, therefore, may affect cellular dopamine release, providing a potential pathogenic mechanism for the DYT1-dependent dystonia.

The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling

“…The formation of membranous whorls caused by expression of ⌬E-torsinA is evident in E-cadherin-positive and SH-SY5Y transfected cells (7,14) but has been also observed in other non-neuronal cell lines overexpressing ⌬E-torsinA (data not shown). These inclusions appear to originate from the NE (47) and have been detected in DYT1 brain, supporting a role in the pathogenesis of dystonia (15).…”

“…Cell Culture-SH-SY5Y cell lines stably transfected with pcDNA3.1, containing either wild type DYT1, GAG-deleted DYT1 (DYT1-⌬E) or no insert (14) were grown in 1:1 mixture of Eagle's minimal essential medium (Promochem, Middlesex, UK), Ham's F-12 nutrient mixture (Invitrogen) and 10% fetal calf serum at 37°C and 5% CO 2 under selective conditions (0.4 mg/ml G418; Invitrogen).…”

“…TorsinA has also been found in the lumen of the endoplasmic reticulum (ER) 3 and in the space between the inner and the outer membrane of the nuclear envelope (NE) (8 -11). In contrast, in cells overexpressing the mutant (⌬E-torsinA), the protein is redistributed from ER to NE and accumulates in large perinuclear membranous inclusions, which appeared to arise from the nuclear envelope (7,(12)(13)(14). TorsinA-positive inclusions have been found in the midbrain of DYT1 patients, suggesting that they are relevant to the pathogenesis of DYT1 dystonia (15).…”

Abnormal motor function and dopamine neurotransmission in DYT1 ΔGAG transgenic mice

“…The increased DA turnover identified in hMT1 mice is noteworthy since tetrabenazine, a dopamine-depleting drug, has been used successfully in patients with generalized dystonia (Jankovic and Orman, 1988). Furthermore, mutant torsinA appears to interfere with the dopamine and vesicular monoamine transporters (Torres et al, 2004;Cao et al, 2005;Misbahuddin et al, 2005). Thus, the hMT1 mouse may be used to study the systems and cellular biology of torsinA and evaluate potential therapeutics for DYT1 dystonia.…”

Commentary: Dopaminergic dysfunction in DYT1 dystonia

“…Within neurons, wild-type torsinA localizes to the endoplasmic reticulum (Liu et al, 2003;Naismith et al, 2004;Hewett et al, 2007), while mutant torsinA protein is found in the perinuclear space (Hewett et al, 2000;Gonzalez-Alegre and Paulson, 2004;Goodchild and Dauer, 2004). Perhaps relevant to the topic of dopaminergic dysfunction to DYT1 dystonia, associations of mutant torsinA with the vesicular monoamine transporter (VMAT2) and with alphasynuclein have been described (Sharma et al, 2001;Misbahuddin et al, 2005).…”