Elizabeth J. Slow

Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration Due to Mutant Huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntingtons disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Cognitive dysfunction precedes neuropathology and motor abnormalities in the YAC128 mouse model of Huntington's disease.

Huntingtons disease (HD) is an adult-onset neurodegenerative disorder involving motor dysfunction, cognitive deficits, and psychiatric disturbances that result from underlying striatal and cortical dysfunction and neuropathology. The YAC128 mouse model of HD reproduces both the motor deficits and selective degeneration observed in the human disease. However, the presence of cognitive impairment in this model has not been determined. Here, we report mild cognitive deficits in YAC128 mice that precede motor onset and progressively worsen with age. Rotarod testing revealed a motor learning deficit at 2 months of age that progresses such that by 12 months of age, untrained YAC128 mice are unable to learn the rotarod task. Additional support for cognitive dysfunction is evident in a simple swimming test in which YAC128 mice take longer to find the platform than wild-type (WT) controls beginning at 8 months of age. YAC128 mice also have deficits in open-field habituation and in a swimming T-maze test at this age. Strikingly, in the reversal phase of the swimming T-maze test, YAC128 mice take twice as long as WT mice to locate the platform, indicating a difficulty in changing strategy. At 12 months of age, YAC128 mice show decreased prepulse inhibition and habituation to acoustic startle. The clear pattern of cognitive dysfunction in YAC128 mice is similar to the symptoms and progression of cognitive deficits in human HD and provides both the opportunity to examine the relationship between cognitive dysfunction, motor impairment, and neuropathology in HD and to assess whether potential therapies for HD can restore cognitive function.

Differential susceptibility to excitotoxic stress in YAC128 mouse models of Huntington disease between initiation and progression of disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG tract in the HD gene. Polyglutamine expansion of huntingtin (htt) results in early, progressive loss of medium spiny striatal neurons, as well as cortical neurons that project to the striatum. Excitotoxicity has been postulated to play a key role in the selective vulnerability of striatal neurons in HD. Early excitotoxic neuropathological changes observed in human HD brain include increased quinolinate (QUIN) concurrent with proliferative changes such as increased spine density and dendritic length. In later stages of the disease, degenerative-type changes are apparent, such as loss of dendritic arborization, a reduction in spine density and reduced levels of 3-hydroxykynurenine and QUIN. It is currently unknown whether sensitivity to excitotoxic stress varies between initiation and progression of disease. Here, we have assessed the excitotoxic phenotype in the YAC128 mouse model of HD by examining the response to excitotoxic stress at different stages of disease. Our results demonstrate that YAC128 mice display enhanced sensitivity to NMDA ex vivo and QUIN in vivo before obvious phenotypic changes. In contrast, 10-month-old symptomatic YAC128 mice are resistant to QUIN-induced neurotoxicity. These findings are paralleled by a significant increase in NMDAR-mediated membrane currents in presymptomatic YAC128 dissociated medium spiny neurons progressing to reduced NMDAR-mediated membrane currents with disease progression. These data highlight the dynamic nature of the mutant htt-mediated excitotoxic phenotype and suggests that therapeutic approaches to HD may need to be altered, depending on the stage and development of the disease.

Full length mutant huntingtin is required for altered Ca2+ signaling and apoptosis of striatal neurons in the YAC mouse model of Huntington's disease

Huntingtons disease (HD) is caused by a progressive loss of striatal medium spiny neurons (MSN). The molecular trigger of HD is a polyglutamine expansion in the Huntingtin protein (Htt). The mutant Htt protein forms insoluble nuclear aggregates which have been proposed to play a key role in causing neuronal cell death in HD. Other lines of investigation suggest that expression of mutant Htt facilitates activity of the NR2B subtype of NMDA receptors and the type 1 inositol 1,4,5-trisphosphate receptors (InsP(3)R1), and that disturbed calcium (Ca(2+)) signaling causes apoptosis of MSNs in HD. The YAC128 transgenic HD mouse model expresses the full-length human Htt protein with 120Q CAG repeat expansion and displays an age-dependent loss of striatal neurons as seen in human HD brain. In contrast, the shortstop mice express an amino-terminal fragment of the mutant Htt protein (exons 1 and 2) and display no behavioral abnormalities or striatal neurodegeneration despite widespread formation of neuronal inclusions. Here we compared Ca(2+) signals in primary MSN neuronal cultures derived from YAC128 and shortstop mice to their wild-type non-transgenic littermates. Repetitive application of glutamate results in supranormal Ca(2+) responses in YAC128 MSNs, but not in shortstop MSNs. In addition, while currents mediated by the NR2B subtype of NMDA receptors were increased in YAC128 MSNs, currents in SS MSNs were found to be similar to WT. Furthermore, YAC128 MSNs were sensitized to glutamate-induced apoptosis. Consistent with these findings, we found that application of glutamate induced rapid loss of mitochondrial membrane potential in YAC128 MSNs. In contrast, SS MSNs do not show increased cell death postglutamate treatment nor accelerated loss of mitochondrial membrane potential following glutamate stimulation. Glutamate-induced loss of mitochondrial membrane potential in YAC128 MSNs could be prevented by inhibitors of NR2B NMDA receptors and mGluR1/5 receptors. Our results are consistent with the hypothesis that disturbed neuronal Ca(2+) signaling plays a significant role in the degeneration of MSN containing full-length mutant Htt(exp). Furthermore, the results obtained with neurons from shortstop mice provide additional evidence that not all fragments of mutant Htt(exp) are toxic to neurons.

Levels of mutant huntingtin influence the phenotypic severity of Huntington disease in YAC128 mouse models

Huntington disease (HD) is a devastating neuropsychiatric disease caused by expansion of a trinucleotide repeat (CAG) in the HD gene. Neuropathological changes include the appearance of N-terminal huntingtin fragments, decreased brain weight and apoptotic neuronal loss in a select subset of neurons located in the striatum. There is still controversy over whether homozygosity for the mutation in HD is associated with a more severe phenotype. In humans, resolution of this issue has been complicated by the small number of homozygous patients and difficulty in the definition of reliable phenotypic endpoints. In order to definitively determine whether there is a correlation between phenotypic severity and expression levels of mutant huntingtin, we undertook a behavioral and neuropathological assessment of YAC128 mice with varying levels of mutant huntingtin. The results reveal a clear relationship between levels of mutant huntingtin and phenotype defined by earlier age of onset, more rapid progression, enhanced striatal volume loss, acceleration of nuclear huntingtin fragment accumulation and increased sensitivity to NMDAR-mediated excitotoxicity. These results provide clear evidence in vivo supporting a more severe phenotype associated with increased levels of mutant huntingtin as seen in homozygotes for HD.

Glutamate receptor abnormalities in the YAC128 transgenic mouse model of Huntington's disease.

A yeast artificial chromosome (YAC) mouse model of Huntingtons disease (YAC128) develops motor abnormalities, age-dependent striatal atrophy and neuronal loss. Alteration of neurotransmitter receptors, particularly glutamate and dopamine receptors, is a pathological hallmark of Huntingtons disease. We therefore analyzed neurotransmitter receptors in symptomatic YAC128 Huntingtons disease mice. We found significant increases in N-methyl-d-aspartate, AMPA and metabotropic glutamate receptor binding, which were not due to increases in receptor subunit mRNA expression levels. Subcellular fractionation analysis revealed increased levels of glutamate receptor subunits in synaptic membrane fractions from YAC128 mice. We found no changes in dopamine, GABA or adenosine receptor binding, nor did we see alterations in dopamine D1, D2 or adenosine A2a receptor mRNA levels. The receptor abnormalities in YAC128 transgenic mice thus appear limited to glutamate receptors. We also found a significant decrease in preproenkephalin mRNA in the striatum of YAC128 mice, which contrasts with the lack of change in levels of mRNA encoding neurotransmitter receptors. Taken together, the abnormal and selective increases in glutamate receptor subunit expression and binding are not due to increases in receptor subunit expression and may exert detrimental effects. The decrease in preproenkephalin mRNA suggests a selective transcriptional deficit, as opposed to neuronal loss, and could additionally contribute to the abnormal motor symptoms in YAC128 mice.

The hunt for BRCA2 interactors scores a big hit: EMSY as a prognostic tool for sporadic breast cancer

The breast cancer susceptibility genes BRCA1 and BRCA2 were first identified over 7 years ago, and while subsequent research has suggested a variety of functions for the proteins encoded by these genes, from DNA repair to transcriptional activation, many questions remain. BRCA1 and BRCA2 do not resemble each other or any other genes in the human genome. Mutations in BRCA1 and BRCA2 that predispose individuals to cancer are commonly loss of function in one allele, and mutations in either of these genes underlie most familial breast cancer cases. However, familial breast cancer only accounts for 5% of breast cancer cases, and somatic mutations in BRCA1 and BRCA2 rarely occur in sporadic breast cancer. In an effort to further investigate the function of BRCA2, the authors performed yeast twohybrid screens to identify interacting proteins. A screen with the N-terminus of BRCA2 yielded 16 interactors, all fragments of the same cDNA. The protein encoded by the cDNA was given the name EMSY after a breast cancer nurse and sister of the first author. The interaction between BRCA2 and EMSY was confirmed, and the region of interaction was delimited to 12 amino acids within a highly conserved transactivation domain at the N-terminus of BRCA2. A reporter assay demonstrated that EMSY could repress the transcriptional activity of BRCA2. Immunofluorescence studies with an antiEMSY antibody revealed that EMSY localizes to the nucleus. The authors treated the cells with ionizing radiation to cause DNA damage and discovered that markers of DNA repair almost completely co-localized with EMSY, indicating a potential role for EMSY in DNA repair processes. When sequence results revealed no similar proteins in the genome, the authors performed twohybrid analysis with the N-terminus of EMSY to discover more about the possible function of EMSY. The Royal Family domain was present in over 80% of the proteins encoded by the interacting clones. The Royal Family domains recognize methylated lysines within histones and may play a role in chromatin regulation. The interaction of EMSY and the proteins HP1 and BS69 (both containing Royal Family domains) was confirmed. These results reveal that EMSY binds to proteins containing Royal Family domains, suggesting that EMSY might play a role in co-coordinating the function of Royal Family domain proteins and therefore chromatin regulation. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping localized EMSY to chromosome 11q13.4–5, an amplification hotspot in sporadic breast cancer. This amplification hotspot is in a gene-rich region with at least four distinct amplification cores. Cyclin D1, a gene found amplified in 20% of breast cancers, is believed to define one of these amplification cores while EMSY is a candidate for another. EMSY is in close proximity to an amplification core previously defined by the GARP gene. In order to determine if EMSY was amplified in breast cancer, the authors used FISH to examine 28 different breast cancer cell lines and cells from five newly diagnosed sporadic breast cancer cases. EMSY was amplified in 18% of the breast cancer cell lines and highly amplified in one of the newly diagnosed cases. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that EMSY is overexpressed in the cell lines exhibiting amplification. Comparative genomic hybridization further revealed that in at least one of the cell lines, the EMSY/GARP amplicon was independently amplified, since other amplification cores in the region remained at normal levels. Expression analysis revealed sixfold overexpression of EMSY in the cell line and barely detectable expression of GARP. These results were confirmed in five other cell Clin Genet 2004: 65: 261–266 Copyright # Blackwell Munksgaard 2004 Printed inDenmark. All rights reserved CLINICALGENETICS doi: 10.1111/j.1399-0004.2004.00240.x

Looping out links Rett syndrome with loss of imprinting error.

Rett syndrome (RTT) is an X-linked dominant disorder, which first presents in females between 6 and 18months of age. After a period of normal development, patients with RTT exhibit clinical features including hand wringing, autism, seizures and later progression to scoliosis and dystonia. Mutations in the gene encoding methyl-CpG binding protein 2 (Mecp2) have been identified in the majority of patients with RTT with 99% of these mutations occurring de novo. Mecp2 is thought to play a role in gene silencing through methylation of CpG islands and through interaction with Sin3A and recruitment of histone deacetylase (Hdac). Methylation and histone modification are thought to be crucial mechanisms for appropriate gene silencing, tissue-specific expression and developmental reprogramming. The symptoms of RTT suggest that Mecp2 plays an important role in postnatal brain development. The neurological phenotypes of the Mecp2-null mice and mice with clinically relevant mutations in Mecp2 are similar to those of RTT, supporting this theory. Microarray studies in Mecp2-null mouse brain studies did not support the prediction that Mecp2 would act as a global gene repressor. The authors of the current study investigated whether Mecp2 could be involved in parental allele-specific methylation or imprinting by repressing transcription of only one allele of an imprinted gene. Errors in imprinting are implicated in Prader-Willi, Angelman and BeckwithWiedemann syndrome. The authors used a chromatin immunoprecipitation (ChIP) assay to isolate genomic DNA sequences which bound Mecp2 in mouse brain. Two genomic Mecp2-binding sites, named Mecp2-1 and Mecp2-2, were localized to mouse proximal chromosome 6, a region homologous to human chromosome 7q21–22. This 3.5 Mbp region includes the imprinted genes Calcr, Peg10, Asb4, DLX5 and the gene Dlx6, the imprinting status of which is unknown. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that transcription of Dlx5 and Dlx6 was approximately two times higher in Mecp2-null mice compared with wildtype (WT) mice. DLX5 in mice is expressed biallelically, although preferentially from the maternal allele. In Mecp2-null mice, however, this preferential expression was lost. DLX5 is expressed specifically from the maternal allele in humans in both brain and lymphoblasts. To investigate whether imprinting of DLX5 is affected in patients with RTT, the authors utilized a polymorphism in Dlx5 to assess allele-specific expression. Of the four RTT lymphoblastoid cell lines (LCLs) identified, three demonstrated biallelic instead of maternalspecific expression of DLX5. Further analysis of each LCL revealed that some, but not all, RTT mutations lead to loss of imprinting of DLX5. To understand the role Mecp2 played in imprinting at the Dlx5 locus, the authors investigated the differentially methylated region (DMR), a possible imprinting control region within mouse proximal chromosome 6. Methylation of the DMR and also the CpG islands lying 50-to Dlx5 and Dlx6 were similar in WT and Mecp2-null mice. High-resolution ChIP was used to discover a third Mecp2-binding region (Mecp2-3) in an intron of Dlx6. CpG islands at this site and nearMecp2-2 were not differentially methylated in WT compared with Mecp2-null mouse. Mecp2, therefore, did not seem to regulate imprinting of Dlx5 through methylation of regional CpG islands. Because Mecp2 associates with Hdacs and histone methyltransferases, two classes of enzymes involved in chromatin silencing, the authors investigated whether Mecp2 was involved in histone-associated silencing of Dlx5. ChIP analysis revealed that the main Hdac1-binding site coincided with Mecp2-3 in WT mouse brain, and Hdac1 binding was almost completely abolished in Mecp2-null mice, indicating the Mecp2 is necessary for Hdac1 binding in this region. Acetylation of histone H3 is associated with transcriptionally active chromatin, while methylation is associated with transcriptionally silent chromatin. Acetylation of histone H3 in the region of the Clin Genet 2005: 67: 391–395 Copyright # Blackwell Munksgaard 2005 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2005.00430.x

Discoveries in Charcot–Marie–Tooth disease, Crohn's disease and Bardet–Biedl syndrome 4

Charcot–Marie–Tooth disease (CMT) is the most common heritable disorder of the peripheral nerves with an estimated prevalence of 1 in 2500. The disorder affects both children and adults and results in slowly progressive neuromuscular impairment. The clinical presentation of the disorder is variable, but most frequently includes atrophy and weakness of distal muscles, particularly of the lower limbs. Patients often require assistance walking or may be unable to walk. Deep tendon reflexes are hypoactive or absent and sensory abilities may be mildly impaired. CMT is also referred to as hereditary motor and sensory neuropathy in the clinical literature or as peroneal muscular atrophy due to the fact that the peroneal nerves are frequently the earliest of the severely affected nerves. Previously, CMT was subdivided into two major groups, the myelinopathies (CMT1) and axonopathies (CMT2), based on electrophysiological and pathological criteria. Insights into the genetic basis for CMT has allowed for further classification of the neuropathy. There are several forms of heritable CMT1, which affect the myelination of peripheral nerves and therefore slow the motor nerve conduction velocity. CMT1A–D are autosomal dominant, CMT1X is X-linked, while CMT1AR A–F (also known as CMT4) are known to be inherited in a recessive fashion. The CMT2 disorders are axonopathies, which do not affect the myelination of peripheral nerves and can also be divided into autosomal dominant (CMT2A–G), X-linked (CMT2X), or recessive (CMT2AR A–B) forms. The clinical phenotype of CMT2 is more variable and less frequent than CMT1 making it difficult to diagnose. CMT2 can be distinguished by normal or only slightly reduced conduction velocity in the peripheral nerves and axonal loss without evidence of demyelination or hypertrophic changes. The inheritance of CMT2A is autosomal dominant and an initial linkage study of six families identified a region in the short arm of chromosome 1 (1p36–p35). In the present study, Zhao et al. happened upon a phenotype strikingly similar to CMT2 in the process of characterizing a microtubule motor protein knockout mouse. Considering the kinesin motor protein, KIF1B, maps to the CMT2A region on chromosome 1 in humans, the authors characterized the KIF1B mice and pursued the hypothesis that KIF1B could be implicated in human CMT2A. Following the initial observation that genetargeted KIF1B+/− mice suffer from a progressive neuropathy similar to the human condition, the authors screened a well-documented CMT2A Japanese pedigree for polymorphisms in the KIF1B gene. The 47 exons of the human KIF1B gene were amplified by PCR and analyzed by SSCP or direct sequencing. Many single nucleotide polymorphisms (SNPs) were detected, but only one, a heterozygous SNP found on exon 3, correlated perfectly with the clinical manifestation of CMT2A. In this pedigree, all four affected individuals have a Q98 to L transformation as a result of an A T point mutation. This polymorphism was not detected in the eight unaffected individuals of this pedigree and 95 unrelated healthy Japanese controls. In this Japanese pedigree, a Q98L missense mutation in the kinesin motor protein KIF1B was identified as the genetic basis of CMT2A.