Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases.

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.

Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73.

Transcriptional disturbance is implicated in the pathology of polyglutamine diseases, including Huntington's disease (HD). However, it is unknown whether transcriptional repression leads to neuronal death or what forms that death might take. We found transcriptional repression-induced atypical death (TRIAD) of neurons to be distinct from apoptosis, necrosis, or autophagy. The progression of TRIAD was extremely slow in comparison with other types of cell death. Gene expression profiling revealed the reduction of full-length yes-associated protein (YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD. Furthermore, novel neuron-specific YAP isoforms (YAPDeltaCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPDeltaCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPDeltaCs also markedly attenuated Htt-induced neuronal death in primary neuron and Drosophila melanogaster models. Collectively, transcriptional repression induces a novel prototype of neuronal death associated with the changes of YAP isoforms and p73, which might be relevant to the HD pathology.

PQBP-1 is expressed predominantly in the central nervous system during development.

Mutations of PQBP-1 (polyglutamine binding protein-1) have been shown recently to cause human mental retardation accompanied by microcephaly at a high frequency. As a first step towards understanding the molecular basis of this developmental anomaly, we analysed developmental expression of PQBP-1 by in situ hybridization, immunohistochemistry and Western blot analysis. Although it had been shown by Northern blot analysis that PQBP-1 mRNA is expressed in multiple organs in adult mice, our present results revealed that PQBP-1 mRNA and protein are dominantly expressed in the central nervous system (CNS) in embryos and in newborn mice. The mean expression level of PQBP-1 reaches a peak around birth and is down-regulated in adulthood. Furthermore, the expression pattern in the CNS changes remarkably following birth. PQBP-1 mRNA in the cerebral cortex is high in embryos but it rapidly decreases after birth. PQBP-1 mRNA increases in external and internal granular cell layers of the cerebellum from postnatal day 1 (P1) to P5. In addition, expression in the subventricular zone, where neurogenesis occurs, was high from P5 to adulthood. Collectively, these findings suggest that PQBP-1 might be involved in neuronal proliferation and/or maturation. These ideas may be relevant to the insufficient growth of brain structure reported in PQBP-1-linked human mental retardation.

Polyglutamine tract-binding protein-1 dysfunction induces cell death of neurons through mitochondrial stress.

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts and colocalizes with mutant polyglutamine proteins. We previously reported that PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype and cell death of motor neurons analogous to human neurodegeneration. To investigate the molecular mechanisms underlying the motor neuron death, we performed microarray analyses using the anterior horn tissues of the spinal cord and compared gene expression profiles between pre-symptomatic transgenic and age-matched control mice. Surprisingly, half of the spots changed more than 1.5-fold turned out to be genes transcribed from the mitochondrial genome. Northern and western analyses confirmed up-regulation of representative mitochondrial genes, cytochrome c oxidase (COX) subunit 1 and 2. Immunohistochemistry revealed that COX1 and COX2 proteins are increased in spinal motor neurons. Electron microscopic analyses revealed morphological abnormalities of mitochondria in the motor neurons. PQBP-1 overexpression in primary neurons by adenovirus vector induced abnormalities of mitochondrial membrane potential from day 5, while cytochrome c release and caspase 3 activation were observed on day 9. An increase of cell death by PQBP-1 was also confirmed on day 9. Collectively, these results indicate that dysfunction of PQBP-1 induces mitochondrial stress, a key molecular pathomechanism that is shared among human neurodegenerative disorders.