Structure/Function Analysis of the Huntingtin N-terminus Encoded by Htt Exon 1 Using Knock-In Mouse Models

Huntington’s disease (HD) is a neurodegenerative disorder that effects up to 1 in every 10,000 people and it is caused by an expansion of the polyglutamine (polyQ) stretch within the Huntingtin (HTT) protein [1]. The HTT polyQ in mammals is flanked by a highly conserved 17 amino acid N-terminal domain (N17), and a proline-rich region (PRR) [2-4]. The PRR is a binding site for many HTT-interacting proteins [5-7], and the N17 domain regulates several normal HTT functions, including HTT’s ability to associate with membranes and organelles [8, 9].

This study investigates the consequence of deleting mouse Huntingtin’s (Htt’s) N17 domain (ΔN17), deleting a combination of its polyQ stretch and PRR (ΔQP), or deleting all three N-terminal domains (ΔNQP) on normal murine Htt function and in the context of an HD mouse model (Q140). HttΔN17, HttΔQP, and HttΔNQP knock-in mice were generated, and their behavior, autophagy function, and neuropathology were evaluated. Homozygous and hemizygous HttΔN17/ΔN17, HttΔQP/ΔQP, HttΔNQP/ΔNQP, HttΔN17/-, and HttΔQP/- mice were generated at the expected Mendelian frequency. HttΔQP/ΔQP mutants exhibit improvements in motor coordination compared to controls (Htt+/+) while in contrast, HttΔN17/ΔN17 mutants do not exhibit any changes in motor coordination, but they do display variable changes in spatial learning and memory. Neither mutant exhibited any changes in basal autophagy in comparison to controls, but thalamostriatal synapses in the dorsal striatum of 24-month-old HttΔN17/ΔN17 mice were decreased compared to controls. These findings support the hypothesis that Htt’s N17, polyQ, and PRR domains are dispensable for its critical functions during early embryonic development, but are likely more important for Htt functions in CNS development or maintenance.

In order to investigate the effects of the N-terminal domain deletions in the context of an HD mouse model, HttΔN17 and HttΔQP mice were also crossed with Q140 mice to generate Htt140Q/ΔN17 and Htt140Q/ΔQP mice. Htt140Q/ΔN17 and Htt140Q/ΔQP mice demonstrated exacerbated rotarod and activity cage phenotypes, but improvements in spatial learning and memory. Investigation of primary striatal neurons from Htt140Q/ΔN17 and Htt140Q/ΔQP mice exposed a deficit in autophagic flux in comparison to controls that was not observed in primary cortical neurons, suggesting that normal Htt’s functions may be somewhat altered in different regions of the brain. Additionally, Htt140Q/ΔN17 primary striatal and cortical neurons had elevated basal p62 immunostaining. These results suggest that expression of the Htt domain deletions in trans with 140Q-Htt can elicit autophagy phenotypes in cortical and striatal neurons.