Huntington disease (HD) is a progressive, autosomal dominantly inherited, neurodegenerative disease that is characterised by involuntary movements (chorea), cognitive decline and psychiatric manifestations. HD is one of a number of late-onset neurodegenerative diseases caused by expanded glutamine repeats, with a likely similar biochemical basis. It has been suggested that the disease-causing mechanism in Huntington disease (and the other polyglutamine disorders) is the ability of polyglutamine to undergo a conformational change that can lead to the formation of very stable anti-parallel β-sheets; more specifically, amyloid structures. This hypothesis is supported by evidence that polyglutamine forms amyloid-like protein aggregates in vitro, which stain with Congo red (a histological stain for amyloid) and exhibit green birefringence under polarized light. Further support for this hypothesis is provided by evidence of Congo red staining with green birefringence, of purified aggregates from HD brain captured on cellulose acetate filters. This study describes the first finding of amyloid-like inclusion bodies in situ, in HD brain. Inclusions possessing an amyloid-like structure were identified by Congo red staining and polarized-light microscopy in the cortex and striatum of HD brain, but these were absent in both normal control brains and Alzheimer's disease brains. Amyloid-like inclusions were of a similar size to the immunohistochemically-detected neuronal inclusions reported by other investigators, however the frequency of the former was far lower than the latter and the relationship between the two types of inclusion is uncertain. Efforts to purify and characterise the amyloid-forming protein(s) in HD utilised the methodologies used to purify amylin from amyloid plaques in the diabetic pancreas, with a number of refinements. Internal fragments of three different proteins, glial fibrillary acidic protein (GFAP), histone H3 and hypothetical protein FLJ20623 were co-purified with the amyloid component of HD brain based on their increased abundance relative to control brains, and subsequently characterised. The role of these proteins in HD is investigated and discussed. In addition, an ~4285 dalton protein was co-purified with the amyloid component of HD brain; this protein was noticeably absent in control brain and displayed unusual biochemical properties, however an amino-acid sequence could not be determined. Interestingly, a protein of the same approximate size, and displaying similar properties, was purified from poorly soluble protein aggregates formed in polyglutamine-expressing cultured cells. SDS-PAGE and Western analysis of sub-cellular fractions of HD and control brain revealed a novel pattern of proteolysis at the N-terminus of huntingtin in HD relative to control, that is supported by recent literature. Myelin basic protein was demonstrated to co-migrate with an N-terminal huntingtin-positive peptide, with apparent molecular weight of ~20 kDa, using tryptic in-gel digestion and protein sequencing. This N-terminal huntingtin-positive peptide and the ~4285 dalton protein described above were both purified from amyloid-containing fractions and both resisted N-terminal protein sequencing. Due to the aberrant migration of polyglutamine-containing proteins in polyacrylamide gels, a possible identity between these peptides is proposed. Laser capture microscopy and tandem mass spectrometry were investigated as potential methods for the purification and characterisation of amyloid-forming proteins in HD and the further development of these techniques may enable the realisation of these aims in the future. The major contribution of this study has been the finding of amyloid-like inclusions in Huntington disease brain. This finding places HD and by association the other polyglutamine disorders into the category of amyloid diseases, and suggests that strategies to prevent amyloid accumulation, a focus of Alzheimer's Disease research, may be of much wider application to glutamine repeat disorders such as HD or vice versa.
| Identifer | oai:union.ndltd.org:ADTP/276665 |
| Date | January 2002 |
| Creators | McGowan, Daniel P. |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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