Restoration of behavioral abnormalities and histopathology by ARL6IP1 gene transfer. (A) Experimental design for AAV9 gene therapy. Each AAV9 vector was delivered to Arl6ip1^−/− mice with 1 μl AAV9 virus (2 × 10¹³ Vg/ml) per primary motor cortex (M1), with two injection sites/mouse (n = 20 mice/group). (B) Representative image of improvement of abnormal limb reflex in Arl6ip1^−/− mice after AAV9 gene delivery (left). Quantification of hindlimb clasping score in Arl6ip1^−/− mice after AAV9 gene delivery. Data represent mean ± SD of n = 18/group and are calculated in GraphPad Prism 7.0 (right). (C) Representative image (left) and measurement of foot-base angle (right) of Arl6ip1^−/− mice after AAV9 gene delivery. Arl6ip1^+/+ mice of the same age were used as positive controls for behavioral analysis. Data represent mean values of n = 18/group ± SD. (D) Representative image showing improvement in disturbed footprint pattern in Arl6ip1^−/− mice after AAV9 gene delivery (left). Stride, stance, and sway length were calculated from footprint patterns of Arl6ip1^−/− mice (right). Data represent mean ± SD of n = 8 mice/group. (E) Mouse coronal section image from Allen Adult Mouse Brain Atlas showing primary motor cortex and hippocampal area (left, marked in purple). Immunofluorescence staining of NeuN and GFAP in the cerebral cortex (top) and hippocampal regions (down) of Arl6ip1^−/− mice after AAV9 gene delivery (200×; scale bar: 100 μm). (F) Quantification of immunofluorescence staining. Data represent mean ± SD of n = 5 mice/group and are analyzed using Student’s t test. **P < 0.01. Data represent averages of three independent biological replicates.