Allele-specific silencing of mutant human ataxin-3 mediates robust reduction of the number of ataxin-3 inclusions and preservation of DARPP-32 immunoreactivity in the rat striatum.

<p>A–R) Co-overexpression of MUT ATX3 and various shRNAs (shAtaxWT, n = 8; shAtaxMUT, n = 7 and shGFP, n = 4) in the striatum of adult rats, 2 months post-injection. The vectors encoding the shRNA cassette and the <i>lacZ</i> reporter gene infect an extensive region of the rat striatum, as shown by β-galactosidase immunoreactivity (A, B and C). shAtaxMUT specifically downregulates MUT ATX3, promoting a significant decrease in the number of MUT ATX3-positive aggregates (E and H ), whereas shAtaxWT has almost no effect on MUT ATX3 expression (F and I), as shown by comparison with the results obtained with the mistargeted shGFP (D and G). A major loss of DARPP-32 immunoreactivity is observed in the striatum infected with MUT ATX3 and shAtaxWT (L and O) or shGFP (J and M), whereas minor DARPP-32 is observed in the striatum infected with shAtaxMUT (K and N), this downregulation being limited to the needle track area. P–R) Quantification of the effect of the different shRNAs on the absolute number (P) and mean size/surface* (Q) of MUT ATX3-positive cells (*p<0,05). R) Quantitative analysis of the DARPP-32-depleted region in the brains of rats in which the striatum was injected with MUT ATX3 and various shRNAs. The lesion volume in brains infected with shAtaxMUT and MUT ATX3 is much smaller than that in brains infected with shAtaxWT or shGFP, indicative of a neuroprotective effect conferred by the selective shAtaxMUT. Statistical significance was evaluated with Fisher's test. (* the mean size of the objects was estimated taking into account the pixels with a gray-scale level for intensity below the mean value, used as a threshold).