Studies of polyglutamine repeats and their biology in relation to disease

Abstract: Polyglutamine repeat expansions of the CAG/CTG type frequently lead to disease characterized by progressive neuronal dysfunction. These diseases typically begins in mid-life and result in severe neurodegeneration. To a certain extent they present with similar features and probably share a common mechanism of pathogenesis. There is an inverse relationship between the size of the repeat and the age at onset, though the contribution of the repeat size to age at onset varies according to the protein context. Intergenerational instability leads to further expansion and an earlier age at onset in succeeding generations, a phenomenon called anticipation. These polyglutamine tracts are encoded at the DNA level by CAG/CTG trinucleotide repeats. The Repeat Expansion Detection (RED) method allows detection of long trinucleotide repeats anywhere in the genome without prior knowledge of their location. In paper I, our aim was to determine if repeats of sequence motifs other than those known at that time might expand. Five new trinucleotide motifs were found in repeat sequences larger than 180 nucleotides (nt) in one or more of the 200 individuals analyzed: ATG/CAT, CCT/AGG, CTT/AAG, TGG/CCA and GTT/AAC. CAG/CTG is the motif that has most frequently been associated with diseases. In paper 11, the RED method was made more sensitive and reliable for screening for CAG/CTG expansions. Multivariate analysis was used to optimize the procedure and to determine the most influential factors in the amplification reaction and to identify interacting factors. It was possible to obtain up to a 5.5 fold increase in the yield of the reaction after optimization of the protocol. In parallel with the optimization procedure, candidate diseases were analyzed by the RED method for the presence of expanded CAG trinucleotide repeats in the genome of the patients. Anticipation appeared to characterize familial spastic paraplegia (FSP) linked to the SPG4 locus (2p21-p24), and an association between expanded CAG/CTG repeats and FSP had been reported. In paper 111, 41 affected individuals from five French and one Polish FSP families linked to SPG4 were tested for expanded CAG/CTG repeats using the RED method, PCR for the ERDA1 and CTG18.1 loci as well as by Western blot for expressed polyglutamines. Our study provided evidence against the hypothesis that a large CAG repeat expansion is the basis of SPG4. This has now been confirmed by the cloning of the gene. In paper IV, eight spinocerebellar ataxia type 7 (SCA7) families with marked anticipation were analyzed by RED for the presence of CAG repeat expansion. A 150-240nt CAG repeat was found to cosegregate with the disease (p<0.000001, n=66). Shortly thereafter, the SCA7 gene was cloned, which opened an avenue for pathophysiological studies of SCAT In paper V, normal ataxin-7 (the SCA-7 gene product) was found to be widely distributed in normal human postmortem brain. The pattern of expression was not limited to regions affected in SCAT In SCA7 brain, the mean proportion of immunolabelled nuclei was significantly higher in regions with neuronal loss (21%) than in regions that were spared (4%) (t=2.1; p<0.05), suggesting that nuclear localization of ataxin-7 might be associated with dysfunction. In paper V1, SCA7 cell culture models were established in HEK293 and SH-SY5Y cells, in order to analyse alterations induced by the mutant ataxin-7 and to compare them with observations in human SCA7 brain. Overexpressed ataxin-7 formed large fibrillar inclusions. It also localized to small electron dense structures that might correspond to nuclear bodies and may possibly be precursors of inclusions. There were indirect signs of ongoing abnormal protein folding, including the recruitment of heat shock proteins and proteasome subunits. Occasionally, transcription factors and polyglutamine containing proteins, including the normal forms of ataxin7 and ataxin-3 were recruited into the inclusions. Our results illustrate for the first time the presence of activated caspase-3 in inclusions in a human polyglutamine disease as well as in the SCA7 cell models. Moreover, caspase-3 expression was up- regulated in cortical neurons, suggesting that it may play a role in the disease process. Finally, on the ultrastructural level, there were signs of autophagy and nuclear indentations, indicative of a major stress response in cells expressing mutant ataxin- 7. The optimised RED technique, should prove useful in identifying additional disorders with trinucleotide repeat expansions as in SCAT The SCA7 cell culture models established in this study share many features of SCA7 brain, that are common to all polyglutamine disorders. However, the observation of activated caspase-3 in inclusions in both cell models and SCA7 brain is particularly interesting. Its role in cell dysfunction and death should be tested in stable models.