Abstract
Nine neurodegenerative diseases, including spinocerebellar ataxia type 7 (SCA7), are caused by the expansion of polyglutamine stretches in the respective disease-causing proteins. A hallmark of these diseases is the aggregation of expanded polyglutamine-containing proteins in nuclear inclusions that also accumulate molecular chaperones and components of the ubiquitin-proteasome system. Manipulation of HSP70 and HSP40 chaperone levels has been shown to suppress aggregates in cellular models, prevent neuronal death in Drosophila, and improve to some extent neurological symptoms in mouse models. An important issue in mammals is the relative expression levels of toxic and putative rescuing proteins. Furthermore, overexpression of both HSP70 and its co-factor HSP40/HDJ2 has never been investigated in mice. We decided to address this question in a SCA7 transgenic mouse model that progressively develops retinopathy, similar to SCA7 patients. To co-express HSP70 and HDJ2 with the polyglutamine protein, in the same cell type, at comparable levels and with the same time course, we generated transgenic mice that express the heat shock proteins specifically in rod photoreceptors. While co-expression of HSP70 with its co-factor HDJ2 efficiently suppressed mutant ataxin-7 aggregation in transfected cells, they did not prevent either neuronal toxicity or aggregate formation in SCA7 mice. Furthermore, nuclear inclusions in SCA7 mice were composed of a cleaved mutant ataxin-7 fragment, whereas they contained the full-length protein in transfected cells. We propose that differences in the aggregation process might account for the different effects of chaperone overexpression in cellular and animal models of polyglutamine diseases.
Highlights
All of these diseases are characterized by continuous accumulation of mutant proteins in insoluble aggregates, typically forming nuclear inclusions (NIs) [6]
These results show that mutant ataxin-7 aggregation can be suppressed in transfected cells by manipulating HSP70 and HDJ2 chaperone expression levels
HSP70/HDJ2 Overexpression Has No Effect on Mutant Ataxin-7 Aggregation in Vivo—Despite the lack of functional improvement in R7E/RH40/RH70 triple transgenic mice, we examined whether mutant ataxin-7 aggregation is modulated by overexpression of these molecular chaperones
Summary
Generation of Transgenic Animals—A 2.2-kb XbaI-SmaI fragment of the human rhodopsin promoter, encompassing the distal enhancer region, was subcloned from the R7N construct [9] into the pGSp2 vector, kindly provided by Dr D. Mohanakumar, using a 3Ј primer encoding a HA epitope These PCR fragments were XhoI-NheI-digested and cloned into the pGS-Rho vector to generate pGS-Rho-RH70 and pGS-Rho-RH40 transgene constructs, respectively. Plasmid Construction and Cell Transfection—For cell transfection experiments, a full-length SCA7 cDNA with 128 CAGs was obtained by PCR amplification of the B7E2B construct [10], using a 5Ј primer encoding a FLAG epitope. This fragment was subcloned into pcDNA3.1(ϩ) (Invitrogen) to generate pc7E2FL. Responses were digitized using a data acquisition labmaster board (Multiliner Vision)
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