Figure 4.

Expression of uN2CpolyG is pathogenic in animals

(A) 60-s tail suspension test shows hind limb clasping in uN2CpolyG-GFP-expressing mice compared with GFP or uN2C-GFP control animals.

(B–D) Time before falling from a rotating rod (B), numbers of paw slips and errors in the notched bar test (C), and maximal distance traveled during 30 min in an open field (D) for AAV2/PHP.eB GFP-injected (n = 6), uN2C-GFP-injected (n = 6), and uN2CpolyG-GFP-injected (n = 11) male mice tested 3 months after injection.

(E) Kaplan-Meier survival curve of AAV2/PHP.eB GFP-injected (n = 6), uN2C-GFP-injected (n = 6), and uN2CpolyG-GFP-injected (n = 8) male mice. Dates of AAV injection and locomotor tests are indicated by arrows.

(F) Immunofluorescence against uN2CpolyG and p62 on cerebellum areas of uN2C-GFP- and uN2CpolyG-GFP-expressing mice sacrificed 2 months after AAV injection. Scale bars, 10 μm.

(G) Quantification of p62- or uN2CpolyG-positive intranuclear inclusions in different brain regions of uN2CpolyG-GFP-expressing mice. Brackets indicate the percentage of co-localization between p62- and uN2polyG-positive intranuclear inclusions. N = 3 mice; at least 200 nuclei were counted per brain region and per animal.

(H) Left panel: immunofluorescence against uN2CpolyG and calbindin on the cerebellum of uN2C-GFP- and uN2CpolyG-GFP-expressing mice sacrificed 4 months after AAV injection. Scale bars, 20 μm. Right panel: quantification of Purkinje cell numbers in GFP-expressing (n = 4), uN2C-GFP-expressing (n = 4), or uN2CpolyG-GFP-expressing (n = 4) mice.

In box-and-whisker plots, box upper and lower limits represent the 25^th and 75^th percentiles, whiskers represent minimum and maximum values, and a horizontal line across the box represents the median. Bar graphs indicate standard error of the mean (SEM). Student’s t test, ^∗∗∗p < 0.001. Nuclei were counterstained with DAPI.

See also Figures S5 and S6 and Videos S1, S2, and S3.