Increased early striatal cell death and HD-like neurochemical changes in Hip14−/− mice. (Ai) No differences in LGE volumes were observed in E12.5, and (Aii) E14.5 Hip14−/− embryos (n= 5). (Aiii) However, a significant decrease in striatal volume was observed at E17.5 (n= 5). (B) Pregnant females were injected with BrdU, and numbers of BrdU-positive cells were assessed in the LGE of (i) day 12.5 in utero (E12.5) and (ii) E14.5 pups. No differences in number of BrdU-positive cells were observed (n= 6). (C) Adjacent sections were stained for TUNEL to assess apoptosis. (i) No difference was observed in the number of TUNEL-positive striatal neurons at E12.5 (n= 6). However, (ii) a significant increase in the number of TUNEL-positive striatal neurons undergoing apoptosis was observed in E14.5 Hip14−/− embryos (n = 7). (D and E) The majority of MSNs express high levels of DARPP32. DARPP32 levels were significantly decreased in 3-month-old Hip14−/− striatum (n = 7). (F and G) Of the DARPP32-containing neurons, the subset expressing enkephalin is predominantly affected in HD. Similarly, enkephalin levels were significantly decreased in the Hip14−/− striatum. (H and I) No changes in substance P levels were observed in the MSNs of Hip14−/− mice (n = 7).

Hip14 is dysfunctional in the presence of mutant HTT. (A) No significant decreases in Htt palmitoylation are observed in Hip14−/− brains compared with the WT (n = 6). (B) HIP14L, a related family member of HIP14, can also act as a PAT for HTT (n= 3). However, (C) HIP14L cannot palmitoylate PSD95 in vitro (n= 5). (D and E) The Hip14 substrates Psd-95 and Snap-25 show significantly reduced palmitoylation in Hip14−/− brains (n= 5). (F) Hip14 protein levels are unaltered in the striatum and cortex from 8-month-old YAC128 mice (n= 3). (G) The palmitoylation of Hip14 is reduced in brains from YAC128 mice indicating a decrease in its ability to act as a PAT (n= 4). (H) Palmitoylation of Snap-25 by Hip14 immunoprecipitated (IP'd) from YAC128 brains is significantly reduced compared with the palmitoylation by Hip14 IP'd from WT mice, indicating Hip14 dysfunction in the presence of mutant HTT (n= 4).

Hip14−/− mice display HD-like neuropathology. (A) The brain weights of Hip14−/− mice were significantly lower than controls (n = 7–11). (B) A predominantly striatal-specific alteration is seen by MRI in Hip14−/− mice (i) shows an axial, and (iii) a sagittal slice from a composite of MRI scans from eight Hip14−/− and eight WT controls at 3 months of age, with (ii) and (iv) showing the same slices with superimposed t-statistics indicating regions of contraction in Hip14−/− mice compared with controls, threshold at a 1% false discovery rate. CPu, caudate/putamen (striatum); cc, corpus callosum; M, motor cortex; LV, lateral ventricle; SC, sensory cortex; H, hippocampus. (C) Quantification of the striatal volume by MRI shows significant reductions in striatal volumes in Hip14−/− mice (n = 8). By immunohistochemistry, significant decreases in striatal volume (D) and (E) neuronal counts are observed in Hip14−/− mice (n = 10–13). (F) No differences in striatal volume are observed in Hip14+/− mice at 12 months (n = 9–11). (G–I) Cortical, hippocampal and white matter volumes are reduced in Hip14−/− brains (n= 8) by MRI. (J) No changes in microglial or astrocyte numbers were observed in the striatum of 3-month-old Hip14−/− mice (n= 7–10).

Loss of excitatory synapses and HD-like behavioral changes in Hip14−/− mice. (A) Representative fields of synapses from electron micrographs of striatum show decreases in the numbers of synapses (arrows) in 3-month-old Hip14−/− mice compared with the WT. (B) Quantification revealed a significant reduction in excitatory but not inhibitory synapses (n= 3 mice per genotype; 40 × 100 µm² fields per genotype). (C) The number of excitatory synapses per neuron is significantly reduced in the Hip14−/− striatum. (D) Electron micrographs showing representative synapses in the striatum from 1-year-old YAC128 and WT mice. As in Hip14−/− mice, a decrease in the total number of synapses is observed. (E) A significant reduction in the number of excitatory synapses is observed in the striatum of YAC128 mice (n = 3 mice per genotype; 40 100 µm² fields per genotype). All values are mean ± SEM. (F) Hip14−/− mice show a significant deficit on the accelerating and (G) fixed rotorod tests, indicating defects in motor co-ordination at 3, 6, 9 and 12 months of age (n = 7–10). In addition, Hip14−/− mice spend a longer time reaching a platform (H) indicating a decrease in swim speed and further implicating motor co-ordination deficits (n= 7–10). (I) Hip14−/− mice are significantly lower in body weight than controls at all ages tested (n= 15). (J–L) Hip14−/− mice are hyperactive as evidenced by increases in stereotypic, horizontal and vertical movements during open-field testing (n= 15–20). (M) Similar to HD patients and YAC128 mice, Hip14−/− mice show significant decreases in PPI compatible with dysfunction in the cortico-striatal pathway (n= 8).