An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

Abstract

Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease.

Figure 1

INT41 reduces Htt-exon1-GFP (Q103) aggregation in HEK293T cells. HEK293T cells were transfected with vectors encoding Htt-exon1-GFP with either a short (Q30) or extended (Q103) glutamine repeat region. Vectors encoding the intrabodies Happ1 or INT41 were cotransfected with Htt-exon1-GFP (Q103). After 40 hours, flow cytometry analysis on a FACS Aria was performed and GFP signals were gated into three categories based on GFP intensity. Htt aggregates are represented in the >100 signal category. The percentage of cells in each category for each treatment is shown.

Figure 2

Time course demonstrating expression and degradation of induced Htt in PC12 cells: (a) PC 12 cells with inducible Htt (either nHtt (Q23) or mHtt (Q73)) were cultured in collagen-coated 12-well plates and induced with Ponasterone A (PonA) or carrier buffer (EtOH). Wells were harvested at the indicated days after induction. Precleared lysates (12 μg) were run in each lane on a 3–8% Tris-Acetate gel, transferred to nitrocellulose, and immunoblotted with rabbit anti-N-terminal Htt antibody and secondary antibody as described (Section 2). Endogenous rat Htt bands are identified by the asterisk (∗). The same membrane was reprobed using an antibody against actin to serve as a loading control. (b) Fold increase of full-length Htt in induced cells compared to vehicle control as determined by ImageJ analysis.

Figure 3

INT41 reduces binding of mHtt fragments to chromatin in PC12 cells: PC12 cells (nHtt Q23 and mHtt Q73) were transduced with rAAV6-INT41 (41) or mock-transduced (M) and then induced for Htt expression after 72 hours with Ponasterone A. Cells were harvested 8 days after induction and then subjected to subcellular fractionation as described (Section 2). 10 μg protein from each fraction was separated on a 3–8% Tris-Acetate gel, then transferred to nitrocellulose, and (a) immunoblotted with rabbit anti-N-terminal Htt antibody and secondary as described (Section 2). (b) Immunoblot with a mouse anti-CREB, anti-GADPH, or anti-Histone H3 as markers for subcellular fractionation. Molecular weights are in kilodaltons as indicated. (c) INT41 distribution into subcellular compartments. A blot from subcellular distribution was reprobed with affinity purified rabbit anti-INT41 to obtain intrabody distribution between cytoplasmic (C), membrane (M), nuclear soluble (NS), and nuclear chromatin (NC) fractions for both mock- (M) and rAAV6-INT41-transduced cells (41). (d) Schematic representation of Caspase 3 and Caspase 6 cleavage of Htt.

Figure 4

INT41 stabilized cognitive function in Huntington's disease R6/2 animal model: mice received bilateral intrastriatal injections of rAAV6-INT41 (R6/2 INT41) or rAAV6-GFP (R6/2 GFP) and wild type mice received vehicle injection (WT VEH) as described in Section 2. At 9-10 weeks, mice were subjected to the T-maze swim test daily for 6–12 days to measure memory acquisition (see Section 2). The ∗ denotes statistical significance of the exhibited cognitive function that is indistinguishable from vehicle-treated control wild type mice as determined by a two-way ANOVA.

Figure 5

INT41 reduced Htt aggregate formation in R6/2 animal model: aggregates in coronal sections of female mice were measured in 4 fields, binned by size, and analyzed for the frequency of sized aggregates as indicated. Analysis of variance on 4113 data points for 0-1 μm size category was 0.085 comparing striatal fields, as described in Section 2, from 5 GFP and 6 INT41 mice.