Molecular analyses of Machado-Joseph disease

Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder with an autosomal dominant inheritance. The brain regions affected by MJD include the Clarke column and spinocerebellar tracts, pons, dentate nuclei, substantia nigra, anterior horn cells, red nuclei, and peripheral nerves, but MJD is distinguished from other inherited cerebellar ataxias because it spares the olives and cerebellar cortex. The established phenotypes of MJD include gait and limb ataxia, ophthalmoplegia, dystonia, amyotrophy, dysarthria, rigidity, pyramidal signs, facial and lingual fasciculations, and bulging eyes (the most characteristic feature).In 1977 Romanul proposed the existence of a new disease entity based on apparent common clinical symptoms in four family pedigrees, including the Machado family and Joseph family (Nakano et al., 1972; Rosenberg et al., 1976). Since these pedigrees were all of Portuguese Azorean origin, the new disease was referred to as “Azorean disease” at the time (Romanul et al., 1997; Nakamoto et al., 1998). Later, the existence of several families with similar clinicopathological phenotypes of non-Azorean origin was reported, and therefore this disease has come to be more preferably known as Machado-Joseph disease (Healton et al., 1980; Lima and Coutinho, 1980; Sakai et al., 1983; Suite et al., 1986; Yuasa et al., 1986; Takiyama et al., 1989).In 1993, a linkage study of large Japanese pedigrees identified the MJD locus on a distal region of the long arm of chromosome 14 (Takiyama et al., 1993). Almost simultaneously, a locus covering a similar region on chromosome 14 was reported as the thirdly identified locus of spinocerebellar ataxia (Stevanin et al., 1994). In this review, we introduce our long-term studies of MJD, covering the identification of MJD1 (the gene responsible for MJD) to the recent molecular analysis of MJD, and from the data obtained from MJD analyses, we discuss the potential general molecular mechanisms that underlie a broad spectrum of neurodegenerative disorders, if not all of them.Neurodegenerative disorders have a considerable range of signs and symptoms: dementia, ataxia, movement disorders, and so forth. However, from a more general point of view, they share several common features, e.g., the inheritances that result in these disorders are usually autosomal dominant, in many cases disease symptoms appear after middle age, and these symptoms progress after appearing. The most prominent pathology is neuronal cell loss and degeneration, although each disorder has its own regions of the central nervous system that are susceptible. Furthermore, for several inherited diseases, such as Huntington’s disease (HD) and dentatorubral-pallidoluysian atrophy (DRPLA), clinical symptoms worsen and the age of onset becomes earlier in each succeeding generation. This phenomenon is called “anticipation.” These features suggest that common mechanisms underlie these inherited neurodegenerative disorders, but the real molecular bases of these mechanisms have long remained unknown.In retrospect, the first breakthrough was made in 1991 by La Spada et al. (1991). They reported the androgen receptor (AR) gene as the gene responsible for spinobulbar muscular atrophy (SBMA), an X-linked recessive neurodegenerative disorder. The gene responsible for HD, the most typical autosomal dominant neurodegenerative disease, was identified in 1993 (The Huntington’s Disease Collaborative Research Group, 1993). Both disorders have totally different symptoms, inheritances, and chromosomal loci (on chromosomes X and 4 in SBMA and HD, respectively), and they both are caused by genes encoding nonhomologous proteins. However, patient-related mutations were expansions (more than 40 repeats) of polyglutamine (polyQ)-coding CAG repeats. The polyglutamines were located near the N-terminal portion in the resulting proteins. Surprisingly, in both diseases, patients with longer expansions appeared to have more severe clinical symptoms and to manifest an earlier disease onset. Even more surprisingly, the highly expanded CAG repeats in HD appeared to be unstably transmitted to succeeding generations, with a tendency toward further elongation, typically in paternal transmission (Duyao et al., 1993). This observation provided a simple but convincing molecular basis of anticipation. Thus, suddenly the possibility was raised that expansions of CAG repeats or other triplet repeats are also responsible for other inherited neurodegenerative disorders, especially those with anticipation.On the assumption that several other inherited neurodegenerative disorders are also caused by CAG triplet expansions in yet unknown genes, we first searched for novel CAG-containing genes that are expressed in the human brain. Using an oligonucleotide with a (CTG)13 repeat as a probe, which we expected to hybridize strongly to CAG repeats and weakly to CGG repeats, we screened a human brain cDNA library. We succeeded in isolating about 30 clones containing CAG repeats, but not CGG repeats (Kawaguchi et al., 1994). One of them consisted of 1,776 bp with a long open reading frame encoding a soluble cytoplasmic protein with an amino acid length of 360 (Kawaguchi et al., 1994) (Fig. 1A). The CAG repeat was predicted to be translated into a glutamine tract, as observed in SBMA and HD, but, instead, was located near the C-terminal portion. Northern blot analysis revealed that transcripts were faintly detectable in all of the tissues examined, with the exception of testis, in which strong expression was observed. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of human brain mRNA demonstrated expression of both alleles with different lengths of CAG repeats, suggesting that each allele had lengths of CAG repeats that were polymorphic in nature. In addition, the originally identified stop codon was later found to be polymorphically changed to a tyrosine-coding TAC in about 60% of the alleles in Japanese (Fig. 1B) (Goto et al., 1997). A splice variant with a different C-terminal end (Fig. 1B) was also identified (Nakamoto et al., 1998).Despite our success with RT-PCR, we were unable to amplify genomic fragments containing the CAG repeats by PCR using any PCR primers that were constructed from the coding sequence. We assumed the presence of an intron close to the CAG repeat and thus screened a human genomic library to isolate the corresponding genomic clones; in the process, we obtained four related genomic fragments. We gave the symbol MJD1 to the gene corresponding to the cDNA and the symbols MJD2, MJD3, and MJD4 to the other three putatively related genes (Kawaguchi et al., 1994). MJD1 and the other three genomic fragments were mapped at 14q32.1, 8q23, 14q21, and Xp22.1, respectively (Kawaguchi et al., 1994). The physiological functions of MJD1 remain to be clarified. However, MJD1 has two ubiquitin-interacting motifs in the N-terminal side of polyQ and carries another motif, named “Josephin,” at the N-terminus (Fig. 1C). A recent report shows that MJD1 is able to bind to HHR23A and HHR23B (the human homologs of the yeast DNA repair protein RAD23) through their ubiquitin-like (UBL) domains (Wang et al., 2000).As predicted, we identified an intron just upstream of the CAG repeat in MJD1. This intron sequence allowed us to amplify by PCR the MJD1 genomic sequence surrounding the CAG repeat. Then, we compared the CAG repeat numbers in MJD1 of healthy individuals with those of clinically diagnosed MJD patients (Fig. 2A) (Kawaguchi et al., 1994). Consistent with our RT-PCR analysis on human brain mRNA, CAG repeat numbers in MJD1 were found to be polymorphic in healthy individuals, from 13 to 36 repeats. The most frequent allele was found to contain 14 repeats, representing about 30% of the total, followed by those with 27, 28, and 21 repeats, representing approximately 15% each. In contrast, MJD patients were found to have longer CAG repeats, from 62 to 84 repeats, and a clear reverse correlation was observed between the repeat numbers and the age of disease onset (Fig. 2B). One repeat elongation was estimated to accelerate onset by approximately 2.6 yr. A recent report shows that rare intermediate repeat lengths, such as 53 and 54 repeats, are also pathogenic (van Alfen et al., 2001). In patients with CAG expansions in both MJD1 genes, onset occurred much earlier than predicted from the repeat numbers, indicating a gene-dosage effect.At almost the same time, an additional two inherited neurodegenerative diseases, spinocerebellar ataxia type 1 (SCA1) and DRPLA, were found to be caused by the expansion of CAG repeats in different responsible genes (Banfi et al., 1993; Koide et al., 1994; Nagafuchi et al., 1994). It was predicted that all of the CAG repeats that had been identified as causing these five inherited neurodegenerations by expansion were transcribed and translated into polyQ stretches as portions of apparent intracellular proteins. Thus, the simplest and easiest idea to test was that expanded polyQ (ex-polyQ) itself plays a toxic function that is causally related to the neurodegeneration.To test this idea, expression vectors for MJD cDNAs with various CAG repeat lengths were transfected in cultured COS7 cells and neuronal PC12 cells, and their phenotypes were (Fig. et al., et al., of these were expressed with a in their were observed by the expression of MJD with and and lengths of were they observed in a C-terminal portion of the MJD protein with a polyQ length cell was the C-terminal portion with and an itself were expressed (Fig. et al., analysis using an that and had suggesting that they in the cells (Fig. et al., In cells with such and of related to observed. The of the and cell were both on the length and of as the protein portions surrounding polyQ the of the and the of cell also or neuronal cell in we in which a similar of MJD and were expressed in cells using the gene et al., of the their and two of were found to have an and gait The affected were which be at a were approximately those of the of their (Fig. et al., The cells were However, we obtained clear of cell In contrast, of the and in the any that the is of causing cell in cultured cell and in In contrast, the MJD those with appeared to have a of if was at and the portions with were and in the on these we a that that or is a in the of the polyQ diseases, that the brain of the with to the of brain regions for each This is able to for the apparent between the affected brain regions and expression of the genes responsible for the polyQ further to the we to cultured cells in which is MJD protein this we expressed in a of cell and these we were unable to any suggesting of the MJD protein in any of the cultured cells examined, which were all However, PC12 cells were several after of we that were made in approximately in a of the transfected PC12 these be with an that an amino acid just of the polyglutamine portion of the MJD and not with an to a to the N-terminus of the MJD protein et al., a cell in which protein is into a strong thus the expression of for This system a novel cell system for isolating cells containing for any protein (Fig. et al., 2001). The protein with a strong at the and with the region of the receptor at the is expressed with a which the expression of a in this for this system in the the MJD in this is by its the portion with to the and the resulting in in PC12 The cells obtained from this system contain PC12 with of the MJD this process, cells with much for the MJD protein be We which a length of polyglutamine repeats in the process, to of the fragments and cell We were able to several PC12 after with and these two and were by and were to have MJD (Fig. et al., 2001). A of the of the MJD based on the of the that the MJD protein is in these cells the amino acid et al., to be not for MJD but also for several other polyQ fragments were found in the in brain of HD, MJD, and as the covering the et al., 1997; et al., et al., et al., it has been reported that an the HD protein in cultured cells, but the responsible to be identified et al., is the of the in polyQ In analysis using and several demonstrated that containing long polyQ surrounding protein portions et al., et al., 1997). In addition, the of the MJD protein was in a to MJD into et al., 1998). These suggest by surrounding portions of the in a of In the of the MJD it a in its N-terminal portion and a the the polyQ which with the observation that the C-terminal portion of MJD to an et al., report that the of MJD is of causing the of and cell suggesting that a of the MJD protein the et al., with cell in to toward cell we constructed PC12 cell polyQ or of the et al., These cells were named and PC12 cells are a cell with the to into cells by the simple of was was from the in this was expressed by the the cells almost all cells after We the of by an analysis using an (Fig. et al., was in the after most cells or more cytoplasmic with many in the large were observed in the as as in the the cells indicating that cytoplasmic and are that cell and cell were not observed in this we found of cells that appeared to be to cell These cells large in the We to these such as simple caused cell These suggest that cell than cytoplasmic and with the that are more observed than cytoplasmic in the affected brain regions of patients with MJD, DRPLA, HD, and other polyglutamine et al., et al., 1997; et al., 1997; et al., these cells, cell were the of cell we the potential of in cell to our of was observed. In addition, several were unable to cell although such as chromosomal were These suggest that were in the of but not in the of cell we the of other and found that protein et al., were after the of cells in the in which was In the cells with a large at was in the that was in with the we found that the protein of had been in the same the cell These suggest the possibility that the of in the a for the Then, recent analysis found that 1 an protein is responsible for in cell et al., at the same time have that many including were into the to the that in gene expression and most typically cell it has been that such as the protein protein and et al., In addition, a different has been proposed as a potential of polyQ disease, in which the of such as are by with et al., et al., is another this that not cell but, function in a et al., 1998). In the of disease, soluble than has been to be toxic to the cell and et al., However, it is not yet known this is to or other are highly in the of MJD patients and in and of Furthermore, it has been reported that several of as as and and are on in the of MJD patients et al., are a common of polyQ These of have raised another possibility that that are in protein and their In cultured cells, have been to in is predicted to result in further of et al., et al., protein has been also reported in other neurodegenerative disorders with cytoplasmic protein et al., et al., et al., These suggest a potential between neuronal and of protein most the the of on cells, especially on we assumed the existence of a a which and the of in the such a potential we a using protein as a of an cultured cells were found to contain a protein with a molecular of approximately that with much more strongly than et al., 2001). This protein was from cells and cells by using analysis and protein revealed that the from both cells were and were a reported protein named a of the Northern blot analysis on human that is expressed not brain but all other tissues as is of the most intracellular and has been to be in in the and et al., et al., et al., 1997; et al., 2001). In these its such as and the using of we which portion of is by the portion was found to be for with (Fig. et al., 2001). analysis of revealed that the portion containing amino acid located near the is responsible for to (Fig. et al., 2001). of three and et al., The and domains are the is and et al., the is predicted to be the to its and et al., We to as a toward as observed by another of the family and et al., 2001). we have not yet succeeded in using cells to this idea, function to to in yeast cells et al., was observed in cells, such as PC12 was found to not with but also with several other protein in neuronal We observed in and cytoplasmic in cell (Fig. et al., 2001). with was also in our and in brain from HD patients (Fig. et al., 2001). Consistent with the idea that a broad range of was found to bind to and the C-terminal portion of containing In addition, was found to with in brain of patients with disease or with (Fig. et al., 2001). also with which were at a by in PC12 cells (Fig. et al., 2001). It is that in these cells with or many large cytoplasmic were followed by cell the same time, we another this time using for genes in We first a of polyQ et al., encoding a and were expressed in the eyes of the of an pathogenic lengths of and cell phenotypes with loss of red loss of and severe of the The phenotypes were on lengths and and were (Fig. et al., to for dominant and of the were with about from a that had chromosomal revealed that as a dominant the gene responsible for this located the region were and two dominant were the and both (Fig. et al., Then, using a the of both were identified to be the region of the Surprisingly, is the of if the is responsible for the of degeneration, we with several such as the strong and loss of function et al., In in allele was (Fig. et al., In was also but much so than in the observed These that the protein plays a function in degeneration, in cell of expression revealed that were in the of the of and in the in were also in et al., (Fig. The and of of both were were with (Fig. et al., of with or to of phenotypes in these but the and of not These that the protein studies by other using similar polyQ disease have that of molecular such as and and is able to in the although the them et al., et al., and In these were also These suggest that the of these in phenotypes are different from those of protein be the This possibility to be was as a that with the MJD protein and was found to bind to an and In addition, were not in but also in in disease and Furthermore, of were identified as dominant for These that is a in neuronal cell by and by other as Thus, we the of from a point of et al., et al., is a with two domains and from yeast to and is for in yeast is called We made a of with amino acid of several amino and as expressed these in different cells, and the of these and the of many large in the the than the et al., et al., acid in both were on those in the A and both in the (Fig. constructed cell or which were referred to as cells and cells, the and we further the phenotypes of these cells (Fig. et al., after both the However, we found clear between the of and cells a healthy and were from the PC12 cells with cells appeared and the to many cytoplasmic which was followed by cell is the most characteristic of Both were into the with the and were also of to such as and It is that made by the system much than than in (Fig. et al., These that of the or that the loss of which many in the followed by cell In to neuronal cell cytoplasmic is another observed in the affected of the polyQ diseases, disease, disease, and other neurodegenerative et al., et al., et al., et al., the origin of these we which and those protein that the of and the so (Fig. et al., in cells, but not in cells, of mRNA was observed (Fig. et al., at a similar to that after a protein et al., another was also in cells, but not in These that the loss or severe of in and Furthermore, in PC12 cells cytoplasmic with as (Fig. et al., suggesting a and protein in the we found an of which are in the and but not in the cytoplasmic of In the such were to (Fig. et al., It is that approximately of in the cells are not and these especially those in are by the in which are into the and by the 1997). Thus, our that in the the of is for into the (Fig. et al., et al., et al., In a et al., was found to more in cells than in cells or These that the of is an in the system and that its severe to and cell neurodegenerative disorders manifest phenotypes movement disorders, and and thus it was to a common molecular if not neurodegenerative However, neurodegenerative disorders share several common such as autosomal dominant and neuronal cell loss and in the affected regions of the central nervous These suggest the existence of similar molecular and neuronal cell many in this have this idea, and a yet unknown toxic has been assumed to be in which typically be observed in inherited with an autosomal dominant of we identified the gene responsible for MJD (Kawaguchi et al., as a this disease has been found to be the most common inherited spinocerebellar ataxia the of Since we have molecular analysis of MJD, to potential common molecular mechanisms MJD but also another inherited neurodegenerative HD, DRPLA, have been to be caused by the expansion of CAG repeats that polyglutamines 1998). we have that polyglutamines have the to and and cell in and and cultured cells, to the of a disease” et al., et al., et al., These of polyglutamines those of and which respectively disease, disease, and Thus, the of is most to be a toxic of function in Then, after the of such have identified a of the not as a protein but also as a cell with our of polyglutamine disease et al., et al., not with polyglutamine but also other protein and which are neuronal typically found in disease and a type of et al., 2001). in its are found to to expansion and cell phenotypes observed in many neurodegenerative et al., These of that functions not as a common for protein but also as a of neurodegenerative of in several neurodegenerative disorders, to is known to function in the in which its is for from the and et al., et al., Even in an approximately to the expression of of such as to of that are most in the and et al., Since functions as a different be in containing from to which in of Furthermore, the of expression of the of in the cells be expected to in to the of recent the of has been to in several of neurodegenerative disorders, including polyglutamine et al., et al., studies have that of by to phenotypes those observed in cells or those observed and of several neurodegenerative disorders et al., 2001). These suggest that of not but also of to different which in to the of the also to different 30% of the are and therefore cells are by a large of that be et al., indicating that cells to the of them and to this at a In to this we the for physiological functions of as as for those related to neurodegenerative of in the is by or is in to the of by the more that are by in the which the in protein in the cells, the its is in the the of the system is in or a of and them in different such as the a the on the is are and their are in the which because of the in by proteins. a the system is and are into the and by the This to protein is However, are as in the of neurodegenerative disorders, this on the to expansion and cell these is by to of followed by cell et al., is and not other It is that is of the most in cells by and to of all and is in many Thus, it is to that not the protein but also other mechanisms protein its the of and them with the of its and with its the molecular mechanisms the physiological of in several neurodegenerative disorders and for the and of such