Deletion of the Huntingtin Proline-Rich Region does not Significantly Affect Normal Huntingtin Function in Mice

Abstract

The N-terminus of Huntingtin, the protein encoded by the Huntington’s disease gene, contains a stretch of polyglutamine residues that is expanded in Huntington’s disease. The polyglutamine stretch is flanked by two conserved protein domains in vertebrates: an N1-17 domain, and a proline-rich region (PRR). The PRR can modulate the structure of the adjacent polyglutamine stretch, and is a binding site for several interacting proteins. To determine the role of the PRR in Huntingtin function, we have generated a knock-in allele of the mouse Huntington’s disease gene homolog that expresses full-length normal huntingtin lacking the PRR. Mice that are homozygous for the huntingtin PRR deletion are born at the normal Mendelian frequency, suggesting that the PRR is not required for essential huntingtin functions during embryonic development. Moreover, adult homozygous mutants did not exhibit any significant differences from wild-type controls in general motor function and motor learning. However, 18 month-old male, but not female, homozygous PRR deletion mutants exhibited deficits in the Morris water task, suggesting that age-dependent spatial learning and memory may be affected in a sex-specific fashion by the huntingtin PRR deletion.

Generation and Characterization of Knock-in Mouse Models Expressing Versions of Huntingtin with Either an N17 or a Combined PolyQ and Proline-Rich Region Deletion

“…Homozygous and hemizygous domain deletion mice ( Htt ΔQP / ΔQP , Htt ΔQP /– , Htt ΔN 17/ ΔN 17 , and Htt ΔN 17/– ) were born at normal Mendelian frequencies in agreement with previous studies, suggesting that the N17, polyQ, and PRR domains are not required for Htt’s essential functions during embryonic development [29, 36, 42, 53]. These results are also consistent with the hypothesis that the N-terminal domains of vertebrate HTT evolved more recently and likely modulate its functions in higher organisms, while the HTT C-terminus contributes to essential functions that are conserved throughout evolution [23].…”

“…Among the 162 mice generated from the Htt ΔN 17/+ intercross, 40 (24.69%) were Htt +/+ , 86 (53.09%) were heterozygous ( Htt ΔN 17/+ ), and 36 (22.22%) were homozygous ( Htt ΔN 17/ ΔN 17 ) (Table 1). Despite the conservation of the N17 domain in all vertebrates, mice homozygous for the N17 deletion ( Htt ΔN 17/ ΔN 17 ) were also born at the normal Mendelian frequency ( χ 2  = 0.815, p  = 0.665, D F  = 3), suggesting that, like the PRR and the polyQ stretch [29, 36], the N17 domain is not required for Htt’s essential functions during embryonic development. Htt ΔQP /+ and Htt ΔN 17/+ mice were also crossed with Htt +/ – mice to determine if absence of normal Htt expression had any effect on the numbers of hemizygous Htt ΔQP /– and Htt ΔN 17/– mice that were obtained.…”

N17 Modifies Mutant Huntingtin Nuclear Pathogenesis and Severity of Disease in HD BAC Transgenic Mice

“…Our study shows that BAC transgenes expressing mutant or WT HTT lacking N17 domain can rescue the embryonic lethality phenotype of murine Htt null mice, suggesting the N17 domain is not required for the essential HTT function during murine development. Our study, together with prior genetic evidence ( Neveklovska et al, 2012 ; Zeng et al, 2010 ) suggests that essential embryonic developmental functions of HTT in vertebrates do not appear to require the three domains near the HTT N-terminus (i.e. the N17, polyQ, and polyproline domains).…”

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

“…Also, transgenic full-length human HTT with expanded polyQ repeats can rescue the embryonic lethal phenotype in mice [ 8 , 44 ]. Deletion of the N-terminal 17 amino acids, polyQ, or the proline-rich domain does not affect mouse development [ 11 , 12 ]. Moreover, the N-terminal region of HTT (<500 amino acids) interacts with a large number of partners [ 45 , 46 ].…”

“…The proline-rich domain (polyP), which follows the polyQ stretch, is found only in mammals. Although polyP may contribute to the solubility of HTT [ 10 ], deletion of polyQ or the proline-rich domain does not affect mouse development [ 11 , 12 ]. N-terminal HTT fragments can be generated by proteolytic processing and other mechanisms.…”