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Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/- mouse models of Huntington's disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/- mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntington's disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington's disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full-length wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/Q111)) huntingtin. We observed that cell death appeared earlier in STHdh(Q111/Q111) cells than in STHdh(Q7/Q7) cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full-length mutant huntingtin in different striatal-derived cells (STHdh(Q7/Q7), M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock-in cells, wild-type cells were transfected with calcineurin A. Calcineurin A-transfected STHdh(Q7/Q7) cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein-transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK-506 produced a more robust reduction in cell death in mutant huntingtin knock-in cells than it did in wild-type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.

Apoptosis, a cell death mechanism regulated by Bcl-2 family members, has been proposed as one of the mechanisms leading to neuronal loss in Huntington's disease (HD). Here we examined the regulation of Bcl-2 family proteins in three different mouse models of HD with exon 1 mutant huntingtin: the R6/1, the R6/1:BDNF+/-, and the Tet/HD94 in which the huntingtin transgene is controlled by the tetracycline-inducible system. Our results disclosed an increase in the levels of the BH3-only proteins Bid and Bim(EL) in the striatum of HD mouse models that was different depending on the stage of the disease. At 16 weeks of age, Bid was similarly enhanced in the striatum of R6/1 and R6/1:BDNF+/- mice, whereas Bim(EL) protein levels were enhanced only in R6/1:BDNF+/- mice. In contrast, at later stages of the disease, both genotypes displayed increased levels of Bid and Bim(EL) proteins. Furthermore, Bax, Bak, Bad, Bcl-2, and Bcl-x(L) proteins were not modified in any of the points analyzed. We next explored the potential reversibility of this phenomenon by analyzing conditional Tet/HD94 mice. Constitutive expression of the transgene resulted in increased levels of Bid and Bim(EL) proteins, and only the Bid protein returned to wild-type levels 5 months after mutant huntingtin shutdown. In conclusion, our results show that enhanced Bid protein levels represent an early mechanism linked to the continuous expression of mutant huntingtin that, together with enhanced Bim(EL), may be a reporter of the progress and severity of neuronal dysfunction., ((c) 2007 Wiley-Liss, Inc.)