HAP40 modulates mutant Huntingtin aggregation and toxicity in Huntington’s disease mice

Huntington’s disease (HD) is a monogenic neurodegenerative disease, caused by the CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene. The HTT gene encodes a large protein known to interact with many proteins. Huntingtin-associated protein 40 (HAP40) is one that shows high binding affinity with HTT and functions to maintain HTT conformation in vitro. However, the potential role of HAP40 in HD pathogenesis remains unknown. In this study, we found that the expression level of HAP40 is in parallel with HTT but inversely correlates with mutant HTT aggregates in mouse brains. Depletion of endogenous HAP40 in the striatum of HD140Q knock-in (KI) mice leads to enhanced mutant HTT aggregation and neuronal loss. Consistently, overexpression of HAP40 in the striatum of HD140Q KI mice reduced mutant HTT aggregation and ameliorated the behavioral deficits. Mechanistically, HAP40 preferentially binds to mutant HTT and promotes Lysine 48-linked ubiquitination of mutant HTT. Our results revealed that HAP40 is an important regulator of HTT protein homeostasis in vivo and hinted at HAP40 as a therapeutic target in HD treatment.

. Western blotting results confirmed the reduction of HAP40 protein in cells transfected with siHap40_613 or siHap40_986.HTT protein expression was also decreased.(B) Immunohistochemistry of the cortex and striatum slices showed that HAP40 is predominately localized in the cytoplasm, and also displays punctate staining in the nucleus (indicated by black arrowheads; 40X, scale bar: 20 μm).(C) Subcellular fractionation of brain tissues was performed to separate nucleus and cytoplasm.Both HAP40 and HTT were predominantly found in the cytoplasm.Histone H3 was used as a nuclear marker, and GAPDH was used as a cytoplasmic marker.

Figure S1 .
Figure S1.HAP40 is mainly localized in the cytoplasm.(A) N2A cells were transfected with two siRNAs targeting Hap40 (siHap40_613 and siHap40_986) or scramble siRNA (Ctrl-siRNA).Western blotting results confirmed the reduction of HAP40 protein in cells transfected with siHap40_613 or siHap40_986.HTT protein expression was also decreased.(B)Immunohistochemistry of the cortex and striatum slices showed that HAP40 is predominately localized in the cytoplasm, and also displays punctate staining in the nucleus (indicated by black arrowheads; 40X, scale bar: 20 μm).(C) Subcellular fractionation of brain tissues was performed to separate nucleus and cytoplasm.Both HAP40 and HTT were predominantly found in the cytoplasm.Histone H3 was used as a nuclear marker, and GAPDH was used as a cytoplasmic marker.
Figure S2.HAP40 is mainly expressed in neurons.(A) Double immunofluorescent staining was performed using cortical slices of WT mice.HAP40 is mainly present in NeuN and β3-tubilin-positive cells, but not in GFAP or F4/80-positive cells (40X, scale bar: 20 μm).(B) Western blotting results show that more HAP40 and HTT are expressed in the grey matter compared the white matter.NeuN was used as a neuronal marker, and GFAP was used as an astrocyte marker.

Figure S7 .
Figure S7.The exogenous HAP40 co-localizes with nuclear mHTT aggregates in the striatum of HD140Q KI mice.(A) Double immunostaining showed that the exogenously expressed HAP40 (detected by HA antibody) colocalizes with nuclear mHTT aggregates (detected by mEM48 antibody) in the striatum of HD140Q KI mice (white arrowheads indicate mHTT aggregates in the nucleus; 40X, scale bar: 20 μm).(B) In HD140Q KI mice, the exogenously expressed HAP40 (detected by HA antibody) is diffused in the cortex but shows punctate patterns in the nucleus of striatal cells.(C) In WT mice, the exogenously expressed HAP40 is diffused both in the cortex and striatum (CTX, cortex; STR, striatum; 40X, scale bar: 20 μm).(D) Western blotting analysis of HTT, HAP40 and NeuN protein expression in the striatum of WT mice injected with AAV-Gfp or AAV-Hap40.Vinculin was used as a loading control.(E) Quantitative analysis of HTT, HAP40, and NeuN protein expression (n = 3, two-tailed student t test; HTT, P = 0.8557; HAP40, P = 0.0072; NeuN, P = 0.5322).Ns, non-significant, **, P < 0.01.Data are presented as mean values ± SEM.

Figure S8 .
Figure S8.The expression of AAV-Hap40 persists seven months after viral injection in the striatum.(A) Western blotting result showing the expression of HAP40 and GFP in both sides of the striatum in 13-monthold HD140Q KI mice injected with either AAV-Hap40 or AAV-Gfp.Vinculin was used as a loading control.(B) Western blotting result of HTT expression in the brain of WT and heterozygous HD140Q KI mice using the D7F7 antibody.(C) Quantitative analysis of wtHTT and mHTT in the brain of heterozygous HD140Q KI mice indicates that D7F7 preferentially reacts with mHTT (n = 4, two-tailed student t test, P = 0.0278).*, P < 0.05.Data are presented as mean values ± SEM.

Figure S9 .
Figure S9.HAP40 reduction does not alter ubiquitination level in WT mice.(A) Western blotting analysis of the total ubiquitination level in the striatum of Cas9 mice injected with AAV-Hap40-gRNA or AAV-Ctrl-gRNA.(B) Quantitative analysis of the total ubiquitin level in the striatum of the virus-injected mice (n = 3, two-tailed student t test, P = 0.09664).(C) Western blotting analysis of HTT expression in WT and 120Q-HEK293 stable cells.(D) HEK293 cells were transfected with three siRNAs targeting HAP40 (siHAP40_959, siHAP40_996 and siHAP40_1073) or scramble siRNA (Ctrl-siRNA).Western blotting results confirmed the reduction of HAP40 in cells transfected with HAP40 siRNAs.(E) Coimmunoprecipitation assay was performed using 120Q-HEK293 cells transfected with the HAP40 siRNA for 48 hours, and then treated with MG132 for another 12 hours.HTT was precipitated by the EPR5526 antibody and K48 ubiquitin antibody was used to detect K48-linked ubiquitination.Reduced K48-ubiquitinated bands in the siHAP40-treated sample are indicated by the bracket.Ns, nonsignificant.Data are presented as mean values ± SEM.