While anti-N-terminal and anti-PRR intrabodies ameliorate the negative effects of mHtt in cell culture and brain slice models of HD, they do so with different efficacy and by different mechanisms. These different mechanisms offer clues to the specific functions of their target domains.
12.3 intrabody was isolated from a yeast surface display library and initially required a 5:1 ratio to mHtt to reduce aggregation (Colby et al., 2004a
). It was then re-engineered, including removal of the disulfide bonds, which do not form in the reducing environment of the mammalian cytoplasm (and can cause mis-folding of intrabodies (Biocca et al., 1995
)), and mutated for greater binding affinity to Htt (Colby et al., 2004b
). In addition to inhibiting mHtt-induced toxicity and aggregation, we find that VL
12.3 also alters cytoplasmic vs. nuclear trafficking of HDx-1.
Modulation of Htt intracellular targeting by the N-terminus has been recently characterized. Removal of this amphipathic alpha helix causes an increase in the level of nuclear Htt, indicating that it functions as a cytoplasmic retention signal (Rockabrand et al., 2007
). Mutation of hydrophobic residues, or the introduction of a helix breaking proline residue in the N-terminal domain results in increased nuclear Htt, suggesting that cytoplasmic retention by the N-terminus is the result of association with organelle and vesicle membranes (Atwal et al., 2007
). Although the N-terminus is not a dimerization domain, disruption of the helical structure also prevents the aggregation of mHtt, which is accompanied by an increase in the toxicity of the protein. Thus, the N-terminus of Htt is required for cytoplasmic localization and the formation of aggregates. The effect on toxicity seen in these experiments may be related to the prevention of aggregation, since mHtt-expressing neurons without aggregates exhibit more toxicity than those with aggregates (Arrasate et al., 2004
). Toxicity related to the N-terminus may also involve altered Htt localization, as the addition of a nuclear localization signal to mHtt increases its toxicity in both cell culture and mouse models of HD (Peters et al., 1999
; Schilling et al., 2004
). Interestingly, while removal or mutation of the N-terminus results in increased toxicity, VL
12.3 binding results in reduced toxicity, suggesting that VL
12.3 may inhibit formation of a toxic conformation or an oligomerization seed molecule. Thus, this intrabody may ameliorate toxicity regardless of mHtt localization or aggregation state.
The polyP and P-rich domains of mHtt are implicated in a number of aberrant protein interactions. These domains are required for mHtt binding to, and sequestering of, several SH3 domain-containing proteins, including proteins associated with vesicle function (Modregger et al., 2002
; Qin et al., 2004
). The PRR of Htt is required for interaction with WW domain-containing proteins (Staub and Rotin, 1996
; Faber et al., 1998
). These include transcription factors, and these interactions are enhanced with increased polyQ repeat length (Passani et al., 2000
; Holbert et al., 2001
). These domains are the site of interaction with IKKγ, a regulatory subunit of the IκB kinase complex. Activation of this complex is known to promote aggregation and nuclear localization of mHtt (Khoshnan et al., 2004
). The PRR of Htt is also the site of P53 interaction and is required for transcriptional repression of P53-regulated genes (Steffan et al., 2000
). Again, this interaction is enhanced by increased polyQ repeat length.
MW7, an intrabody recognizing pure polyP, reduces mHtt-induced aggregation and toxicity in cell culture and in Drosophila
models of HD (Khoshnan et al., 2002
; Jackson et al., 2004
). We find that it is also effective in an acute brain slice model of HD, and that it increases the turnover of HDx-1, with greater effect on the mutant than the wild type form. We also produced novel intrabodies, Happ1 and 3, which recognize the unique, P-rich epitope between the two polyP domains of Htt. The Happ intrabodies exhibit beneficial properties similar to those of MW7 such as preferential effects on the mutant form of Htt and increasing turnover without altering localization, but the Happs are effective at lower ratios to Htt than MW7. We found no evidence that the anti-PRR intrabodies bind to previously aggregated mHtt, suggesting that the observed reduction in aggregation is the indirect result of increased turnover of the soluble form of the protein, causing a shift away from the aggregated state. The increased turnover of HDx-1 in the presence of either anti-polyP or anti-P-rich intrabodies suggests that this effect is a direct result of blocking these epitopes and therefore that this domain has a role in modulating stability of the mutant protein.
Disruption of mHtt stability by Happ binding could have therapeutic potential. The success of RNAi experiments show that reduction of mHtt levels is an effective therapeutic strategy (Harper et al., 2005
; Rodriguez-Lebron et al., 2005
; Machida et al., 2006
). Unlike RNAi however, these intrabodies can distinguish between the wt and mutant forms of Htt, which is preferable, as the loss of normal Htt function can have negative effects (Dragatsis et al., 2000
; Leavitt et al., 2001
; Zuccato et al., 2001
). The ability of the Happ intrabodies to increase turnover of mHtt may ameliorate the disruption of the ubiquitin proteasome seen in HD, although it is presently unclear if this increased turnover occurs through a ubiquitin-dependent pathway. As the levels of intrabody protein are increased in the presence of Htt, it is likely that the intrabodies direct the breakdown of mHtt without themselves being degraded. Moreover, the Happs, although significantly more effective than the original intrabody isolated and matured to become VL
12.3, have yet to undergo any re-engineering and could potentially be improved by removal of disulfide bonds and mutation for greater Htt binding affinity. In addition, the present results with the Happ intrabodies highlight the importance of the unique, P-rich domain in mHtt toxicity.