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Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.

Friedreich's ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.

Mismatch repair (MMR) proteins have critical roles in the maintenance of genomic stability, both class-switch recombination and somatic hypermutation of immunoglobulin genes and disease-associated trinucleotide repeat expansions. In the genetic absence of MMR, certain tissues are predisposed to mutations and cancer. MMR proteins are involved in various functions including protection from replication-associated and non-mitotic mutations, as well as driving programmed and deleterious mutations, including disease-causing trinucleotide repeat expansions. Here we have assessed the levels of MSH2, MSH3, and MSH6 expression in a large number of murine tissues by transcript analysis and simultaneous Western blotting. We observed that MMR expression patterns varied widely between 14 different tissue types, but did not vary with age (13-84 weeks). MMR protein expression is highest in testis, thymus and spleen and lowest in pancreas, quadriceps and heart, with intermediate levels in liver, kidney, intestine, colon, cortex, striatum and cerebellum. By equalizing antibody signal intensity to represent levels found in mMutSα and mMutSβ purified proteins, we observed that mMSH3 protein levels are greater than mMSH6 levels in the multiple tissues analyzed, with more MSH6 in proliferating tissues. In the intestinal epithelium MSH3 and MSH6 are more highly expressed in the proliferative undifferentiated cells of the crypts than in the differentiated villi cells, as reported for MSH2. This finding correlates with the higher level of MMR expression in highly proliferative mouse tissues such as the spleen and thymus. The relative MMR protein expression levels may explain the functional and tissue-specific reliance upon the roles of each MMR protein., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases., Competing Interests: The authors have declared that no competing interests exist.

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

Hyperthermia adversely affects cell structure and function, but also induces adaptive responses that allow cells to tolerate these stressful conditions. For example, heat-induced expression of the molecular chaperone protein HSP70 can prevent stress-induced cell death by inhibiting signaling pathways that lead to apoptosis. In this study, we used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We found that in heat-shocked cells, the actin-severing protein cofilin acquires inhibitory Ser3 phosphorylation, which is associated with an inhibition of chemokine-stimulated cell migration. Cofilin phosphorylation appeared to occur as a result of the heat-induced insolubilization of the cofilin phosphatase slingshot (SSH1-L). Overexpression of HSP70 reduced the extent of SSH1-L insolubilization and accelerated its resolubilization when cells were returned to 37°C after exposure to hyperthermia, resulting in a more rapid dephosphorylation of cofilin. Cells overexpressing HSP70 also had an increased ability to undergo chemotaxis following exposure to hyperthermia. These results identify a critical heat-sensitive target controlling cell migration that is regulated by HSP70 and point to a role for HSP70 in immune cell functions that depend upon the proper control of actin dynamics.

Huntington's disease (HD) and myotonic dystrophy (DM1) are caused by trinucleotide repeat expansions. The repeats show different instability patterns according to the disorder, cell type and developmental stage. Here we studied the behavior of these repeats in DM1- and HD-derived human embryonic stem cells (hESCs) before and after differentiation, and its relationship to the DNA mismatch repair (MMR). The relatively small (CAG)44 HD expansion was stable in undifferentiated and differentiated HD hESCs. In contrast, the DM1 repeat showed instability from the earliest passages onwards in DM1 hESCs with (CTG)250 or (CTG)1800. Upon differentiation the DM1 repeat was stabilized. MMR genes, including hMSH2, hMSH3 and hMSH6 were assessed at the transcript and protein levels in differentiated cells. The coincidence of differentiation-induced down-regulated MMR expression with reduced instability of the long expanded repeats in hESCs is consistent with a known requirement of MMR proteins for repeat instability in transgenic mice. This is the first demonstration of a correlation between altered repeat instability of an endogenous DM1 locus and natural MMR down-regulation, in contrast to the commonly used murine knock-down systems.

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntington's disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSbeta. Higher or lower levels of hMutSbeta or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50*(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSbeta requirement for instability, hMutSbeta is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each other's repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSbeta. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions.

Arylsulfatase-C and sterol sulfatase were thought to be identical enzymes whose X-linked locus escapes inactivation. However, recent evidence shows that they are not identical but that arylsulfatase-C in human fibroblasts exists in two isozymic forms, designated as slow and fast. We now report that the two forms are enzymatically different. When assayed with an artificial fluorogenic substrate, the slow form showed a pH optimum of 8.00 and a Km of 228 microM. In contrast, the fast form showed a pH optimum of 7.67 and a Km of 86.7 microM with substrate inhibition occurring above 0.33 mM. The heat stability of the fast form was slightly below that of the slow form. Polyclonal antibodies raised against the slow form did not cross-react with the fast form. Hence, the two isozymic forms of arylsulfatase-C are enzymatically and structurally different and the slow form is associated with sterol sulfatase activity.

The dynamic changes in skin mast cell (MC) numbers around incised wounds were studied, using experimental designs amenable to multiple analyses of variance. Sixty-four Wistar albino rats were shaved in the interscapular region, wounded or not wounded, and then killed 2 or 10 days later. During this period, the rats were exposed continually to a cold (2 degrees C) or control (20 degrees C) climate and treated daily over the shaved region with either tap water or a weak sulphuric acid (pH 3.5) solution. The MCs within five adjacent fields of the wound or the control reference and within the superficial and deep halves of the skin were counted (at x 400). The greatest decrease in MC numbers occurred within about 700 microns of the wound. Whereas the paucity of MCs within the wound region was evident at 2 days, near-normal levels were achieved by day 10. Cold exposure produced little effect, but MCs responded differently to the water and acid treatments as a function of distance and skin depth.