Transglutaminase and diseases of the central nervous system.

Alzheimer's disease, Parkinson's disease and diseases of expanded polyglutamine are associated with insoluble protein aggregates and neuronal death. A role for transglutaminase in the stabilization of these aggregates has been proposed. Diseases of polyglutamine expansion have been the most thoroughly investigated and a large body of studies supports the causative role of transglutaminase in aggregation of expanded polyglutamine. However none of the evidence is conclusive. Indisputable evidence of cross-linking by transglutaminase will be required in order to provide firm support for therapeutic measures based on the role of transglutaminase

[N(epsilon)-(gamma-glutamyl) lysine] as a potential biomarker in neurological diseases : New detection method and fragmentation pathways.

Protein aggregates are characteristic of a number of diseases of the central nervous system such as diseases of polyQ expansion. Covalent bonds formed by the action of transglutaminase are thought to participate in the stabilization of these aggregates. Transglutaminase catalyzes the formation of cross-links between the side chains of glutaminyl and lysyl residues of polypeptides. Identification of the isodipeptide N(epsilon)-(gamma-glutamyl) lysine (iEK) in terminal proteolytic digests of neuronal aggregates would demonstrate participation of transglutaminase in neurological diseases. In order to identify and quantify the iEK present in the brain of patients with neurological disease, a method combining liquid chromatography and multistep mass spectrometry was developed. Because isobaric peptides of iEK could be present in the digest of aggregated proteins, the choice of fragment diagnostic ions was crucial. These ions were identified by mass spectrometry on sodiated iEK, which was derivatized on the carboxylic functions and terminal amines in order to improve sensitivity. Deuterated molecules as well as (13)C(6)- and (15)N(2)-isotopomers were used to derive filiations in the multistep fragmentations. The main fragmentation patterns have been identified, so that two ions (m/z 396 [MH - 56-42 u](+) and 350 [MH - 56-88 u](+)) are shown to be adequate markers for quantitation experiments. In order to gain a better understanding of the fragmentation processes, detailed quantum chemical calculations have been performed at levels which are expected to provide good accuracy. A thorough study has been carried out with a reduced model in which only the 'active' part of the molecule is retained. This allowed obtaining full mechanistic details on the pathways leading to a number of observed fragments. In particular, it has been shown that losses of 87 and 88 u from A(+) = [MH - 56 u](+) are competitive. Computations on the entire derivatized isodipeptide have been used to validate the use of the smaller model in order to obtain reliable energetics and mechanisms. Copyright (c) 2007 John Wiley & Sons, Ltd

Inclusions of R6/2 Mice Are Not Amyloid and Differ Structurally from Those of Huntington Disease Brain

R6/2 mice contain an N-terminal fragment of human huntingtin with an expanded polyQ and develop a neurological disease resembling Huntington disease. Although the brain of R6/2 mice contains numerous inclusions, there is very little neuronal death. In that respect, R6/2 mice differ from patients with Huntington disease whose striatum and cerebral cortex develop inclusions associated with extensive neuronal loss. We have previously demonstrated using synchrotron-based infrared microspectroscopy that the striatum and the cortex of patients with Huntington disease contained inclusions specifically enriched in amyloid β-sheets. We had concluded that the presence of an amyloid motif conferred toxicity to the inclusions. We demonstrate here by synchrotron based infrared microspectroscopy in transmission and attenuated total reflectance mode that the inclusions of R6/2 mice possess no detectable amyloid and are composed of proteins whose structure is not distinguishable from that of the surrounding soluble proteins. The difference in structure between the inclusions of patients affected by Huntington disease and those of R6/2 mice might explain why the former but not the latter cause neuronal death

Structure of Inclusions of Huntington’s Disease Brain Revealed by Synchrotron Infrared Microspectroscopy: Polymorphism and Relevance to Cytotoxicity

Huntington’s disease is caused by a polyglutamine expansion in huntingtin. Affected brain regions contain characteristic aggregates of the misfolded expanded protein. Studies in cells and animals show that aggregates are polymorphic and that the secondary structure of the aggregates is likely to condition their cytotoxicity. Therefore knowing the structure of aggregates is important as neurotoxic secondary structures may be specifically targeted during the search for prophylactic or therapeutic drugs. The structure of aggregates in the brain of patients is still unknown. Using synchrotron based infrared microspectroscopy we demonstrate that the brains of patients with Huntington disease contain putative oligomers and various kinds of microscopic aggregates (inclusions) that can be distinguished by their differential absorbance at 1627 cm^–1 (amyloid β sheets) and 1639 cm^–1 (β sheets/unordered). We also describe the parallel/antiparallel organization of the β strands. As the inclusions enriched in both β sheets and β sheets/unordered structures are characteristic of severely affected brain regions, we conclude that this kind of amyloid inclusions is likely to be particularly toxic to neurons

Misfolding of Proteins with a Polyglutamine Expansion Is Facilitated by Proteasomal Chaperones*S⃞

Deposition of misfolded proteins with a polyglutamine expansion is a hallmark of Huntington disease and other neurodegenerative disorders. Impairment of the proteolytic function of the proteasome has been reported to be both a cause and a consequence of polyglutamine accumulation. Here we found that the proteasomal chaperones that unfold proteins to be degraded by the proteasome but also have non-proteolytic functions co-localized with huntingtin inclusions both in primary neurons and in Huntington disease patients and formed a complex independently of the proteolytic particle. Overexpression of Rpt4 or Rpt6 facilitated aggregation of mutant huntingtin and ataxin-3 without affecting proteasomal degradation. Conversely, reducing Rpt6 or Rpt4 levels decreased the number of inclusions in primary neurons, indicating that endogenous Rpt4 and Rpt6 facilitate inclusion formation. In vitro reconstitution experiments revealed that purified 19S particles promote mutant huntingtin aggregation. When fused to the ornithine decarboxylase destabilizing sequence, proteins with expanded polyglutamine were efficiently degraded and did not aggregate. We propose that aggregation of proteins with expanded polyglutamine is not a consequence of a proteolytic failure of the 20S proteasome. Rather, aggregation is elicited by chaperone subunits of the 19S particle independently of proteolysis