Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes.

Modifying the length of the Huntington's disease (HD) CAG repeat, the major determinant of age of disease onset, is an attractive therapeutic approach. To explore this we are investigating mechanisms of intergenerational and somatic HD CAG repeat instability. Here, we have crossed HD CAG knock-in mice onto backgrounds deficient in mismatch repair genes, Msh3 and Msh6, to discern the effects on CAG repeat size and disease pathogenesis. We find that different mechanisms predominate in inherited and somatic instability, with Msh6 protecting against intergenerational contractions and Msh3 required both for increasing CAG length and for enhancing an early disease phenotype in striatum. Therefore, attempts to decrease inherited repeat size may entail a full understanding of Msh6 complexes, while attempts to block the age-dependent increases in CAG size in striatal neurons and to slow the disease process will require a full elucidation of Msh3 complexes and their function in CAG repeat instability.

Sorafenib inhibits growth and mitogen-activated protein kinase signaling in malignant peripheral nerve sheath cells.

Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue tumors with a very poor prognosis and largely resistant to chemotherapy. MPNSTs are characterized by activation of the Ras pathway by loss of tumor suppressor neurofibromatosis type 1. In view of this, MPNST may be susceptible to inhibition of the activated Ras/Raf/mitogen-activated protein kinase pathway by the B-Raf inhibitor sorafenib. MPNST (MPNST and ST8814) and dedifferentiated liposarcoma (LS141 and DDLS) human tumor cell lines were characterized for Ras activation and B-Raf expression. Tumor cells were treated with sorafenib and examined for growth inhibition, inhibition of phospho-MEK, phospho-ERK, cell cycle arrest, and changes in cyclin D1 and pRb expression. MPNSTs were sensitive to sorafenib at nanomolar concentrations. This appeared to be due to inhibition of phospho-MEK, phospho-ERK, suppression of cyclin D1, and hypophosphorylation of pRb at the CDK4-specific sites, resulting in a G(1) cell cycle arrest. These effects were not seen in the liposarcoma cells, which either did not express B-Raf or showed decreased Ras activation. Small interfering RNA-mediated depletion of B-Raf in MPNSTs also induced a G(1) cell cycle arrest in these cells, with a marked inhibition of cyclin D1 expression and Rb phosphorylation, whereas depletion of C-Raf did not affect either. With growth inhibition at the low nanomolar range, sorafenib, by inhibiting the mitogen-activated protein kinase pathway, may prove to be a novel therapy for patients with MPNST.

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice.

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.

Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis.

BACKGROUND: JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. RESULTS: To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. CONCLUSIONS: These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.

Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum.

Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in Hdh(Q111) mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of Hdh(Q111/+):Msh2(+/+) and Hdh(Q111/+): Msh2(-/-) progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an approximately 5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.

Cln3(Deltaex7/8) knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth.

Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an approximately 1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3( Deltaex7/8) homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3( Deltaex7/8) mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons.

Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice.

The hallmark striatal neurodegeneration of Huntington's disease (HD) is first triggered by a dominant property of the expanded glutamine tract in mutant huntingtin that increases in severity with glutamine size. Indeed 111-glutamine murine huntingtin leads to a dominant cascade of phenotypes in Hdh(Q111) mice, although these abnormalities are not manifest in Hdh(Q50) mice, with 50-glutamine mutant protein. Therefore, to identify phenotypes that might reflect events closer to the fundamental trigger mechanism, and that can be measured as a consequence of adult-onset HD mutant huntingtin, we have screened for altered expression of genes conserved in evolution, which are likely to encode essential proteins. Probes generated from Hdh(Q111) homozygote and wild-type striatal RNAs were hybridized to human gene segments on filter arrays, disclosing a mutant-specific increase in hybridization to Rrs1, encoding a ribosomal protein. Subsequent, quantitative RT-PCR assays demonstrated increased Rrs1 mRNA from 3 weeks of age in homozygous and heterozygous Hdh(Q111) striatum and increased Rrs1 mRNA expression with a single copy's worth of 50-glutamine mutant huntingtin in Hdh(Q50) striatum. Moreover, quantitative RT-PCR assays for the human homologue demonstrated elevated Rrs1 mRNA in HD compared with control postmortem brain. These findings, therefore, support a chronic impact of mutant huntingtin on an essential ribosomal regulatory gene to be investigated for its role very early in HD pathogenesis.