Huntington disease (HD) is an autosomal dominant neurodegenerative disorder. To investigate the mechanism of neurodegeneration induced by mutant huntingtin, we developed a stable neuro2a cell line expressing truncated N-terminal huntingtin (tNhtt) with EGFP using the ecdysone-inducible system. The formation of aggregates and the cell death induced by expression of tNhtt with expanded polyglutamine was repeat length- and dose-dependent. Caspases were activated, and the death substrates of caspases, lamin B and ICAD (an inhibitor of caspase-activated DNase), were cleaved in this cell death process. The cleavage of lamin B was inhibited by caspase inhibitors. These findings suggest that the cell death induced by tNhtt with expanded polyglutamine is mediated by caspases.

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the downregulation of specific genes in this region of the brain.

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When the human cDNA, isolated on the basis of homology to the murine carbonic anhydrase (CA) "Y" was expressed in COS cells, the human CA was targeted to and processed in mitochondria, as expected for CA-V. However, tissue distribution reported for the corresponding mouse CA Y mRNA was much more limited than that reported for the distribution of CA-V immunostaining in rat tissues. To determine whether the murine cDNA actually encodes a mitochondrial CA activity and to compare the tissue distribution of the homologous murine and rat gene products, we used reverse transcription-PCR to reisolate the murine CA-V candidate cDNA and used the murine cDNA probe to isolate the homologous rat cDNA. We compared the two cDNA sequences, the activities they expressed after transfection of COS cells, and the sites of N-terminal processing of expressed products. In addition, we used antibodies to the C-terminal peptides predicted from each cDNA to compare distribution of CA-V in mouse and rat tissues and to identify CA-Vs in mitochondria isolated from mouse and rat liver. From these studies, we conclude that both mouse and rat CA-V candidate cDNAs encode active CAs that are targeted to and processed in mitochondria and that there are real differences in tissue distribution of CA-V between mouse and rat. However, the findings that are M(r) of CA-V in rat tissues is smaller than that previously reported and that the tissue distribution also differs lead us to conclude that the antibody used in prior reports most likely misidentified another antigen in rat tissues as CA-V.

A full-length cDNA clone encoding human mitochondrial carbonic anhydrase (CA), CA V, was isolated from a human liver cDNA library. The 1123-bp cDNA includes a 55-bp 5' untranlated region, a 915-bp open reading frame, and a 153-bp 3' untranslated region. Expression of the cDNA in COS cells produced active enzyme. The 34-kDa precursor and 30-kDa mature form of CA V were identified on Western blots of COS-cell homogenates by a CA V-speciflc antibody raised to a synthetic peptide corresponding to the C-terminal 17 aa of CA V. Both 34-kDa and 30-kDa bands were also present in mitochondria isolated from trnsfected COS cells, whereas only the 30-kDa band was present in mitochondria isolated from normal human liver. The N-terminal sequence determined directly on the 30-kDa soluble CA purified from transfected COS cells indicated that processing of the precursor to mature human CA V involves removal ofa 38-aa mitochondrial leader sequence. The 267-aa sequence deduced for mature