The spontaneous CAG-repeat elongation observed in R6/2 mice bred via the male germ-line has led to the paradoxical discovery that highly expanded CAG repeats (i.e. >250) show a prolonged disease phenotype, with increased lifespan, as opposed to the expected decreased lifespan. This phenomenon has been observed in several different R6/2 colonies (Cowin et al. 2008
; Cummings et al. 2008
; Thomas et al. 2008
; Dragatsis et al. 2009
; Morton et al. 2009
). The mechanism for this attenuated phenotype is unclear. In the current study, we compared gene expression profiles between R6/2 transgenic mice identical for the human HD transgene (a 1.9 kb genomic fragment containing promoter sequence and exon 1) but with differing CAG repeat lengths, in order to identify genes that may be related to the prolonged phenotype. We find that genes decreased in expression due to the presence of mutant Htt are similar in R6/2 lines carrying standard (~150 CAG) and highly expanded (~300 CAG) repeats, while genes upregulated in their expression are largely distinct to each line. This suggests that those genes upregulated in expression may be important modifiers of disease pathogenesis. In particular, genes found to be upregulated distinctly in R6/2Q300
transgenic mice were associated with protein processing, including establishment of protein localization, protein folding and ubiquitination, including Lrsam1, Nasp, Rab9b, Erp29
which we validated by real-time PCR analysis. These pathways, as they relate to the htt protein levels, have been widely implicated as modifiers of HD pathogenesis (Finkbeiner and Mitra, 2008
Studies on R6/2 mice with highly expanded repeats have revealed potential therapeutic relevance of the role of nuclear inclusions on HD pathology. In all three published reports (including this one) the patterns of htt aggregation in mice with >250 CAG repeats is dramatically different than that observed in mice with ~150 CAG repeats. This pattern directly correlated with disease onset and lifespan. These findings suggest that there is a length-dependency in the mechanisms underlying formation of nuclear inclusions and support the idea that nuclear inclusion formation is deleterious (Steffan and Thompson, 2003). A previous report has proposed that hindrance of nuclear entry in mice with greater than 300 CAG repeats might be due to the protein product becoming too large for passive nuclear entry (Dragatsis et al. 2009
). However, the htt inclusions that are formed do not appear to correspond to the predicted CAG repeat length, as they are no larger than those observed in mice with 150 CAG repeats; rather the cytoplasmic inclusions that we detect in our mice are smaller than those observed in mice with lower CAG repeat sizes (see ). Moreover, a similar inclusion phenotype also correlates with the rescue of HD neurotoxicity in mutant htt-exposed striatal neurons by overexpression of molecular chaperones (Perrin et al. 2007
). Thus, we hypothesize that the increased expression of genes related to protein localization and protein folding affects the processing and destination of mutant htt in the cell. The mechanism by which highly expanded polyglutamine repeats lead to the upregulation of these transcripts is not known; however, it is possible that this reflects a compensatory response by the cell to mobilize and redistribute the toxic protein.
In this study, we further focused on Lrsam1 (a.k.a. Tal), a multifunctional RING finger protein, with E3 ubiquitin ligase activity (Amit et al. 2004
), although the exact functional role(s) for this protein are unclear. Our in vitro
experiments in primary cultured cells demonstrated that the overexpression of Lrsam1 reduces polyQ toxicity. We show that Lrsam1 prevented the loss of primary striatal neurons expressing htt171-82Q and reduced the presence of nuclear htt inclusions. A possible mechanism for this rescue of HD neurotoxicity is the inhibition or reversal of nuclear htt aggregate load. Previous studies have shown that Lrsam1 contributes to the sorting of proteins into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane (Amit et al. 2004
). Hence, increased expression of Lrsam1 might act to shift the localization of mutant htt out of the nucleus and into the cytoplasm, although targeting to specific cytoplasmic organelles could not be delineated in this study. Another gene that was substantially elevated in R6/2Q300
transgenic mice is Erp29, which has been hypothesized to act as a chaperone that may facilitate folding and/or export of secretory proteins from the endoplasmic reticulum (Sargsyan et al. 2002
). Hence, it may act as a chaperone for htt, facilitating its localization in the cytoplasm, although we did not detect any effects of Erp29 alone on htt nuclear inclusions in our in vitro
HD model, suggesting that it may require the cooperation of other proteins.
Overall, we have identified a group of genes that are upregulated in expression in R6/2 transgenic mice with 300 CAG repeats, but not in R6/2 mice with 150 CAG repeats at both middle and late stages of illness, many of which are related to protein processing. We hypothesize that increased expression of these genes, in particular Lrsam1, results in a delayed disease onset, latent disease progression and longer lifespan. Increasing the activity of Lrsam1, and potentially other similar genes in humans with a goal of reducing nuclear inclusion load, may have therapeutic relevance in HD patients.