Neurodegenerative Disorders

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The prevalence of neurodegenerative disorders is growing exponentially in developed countries, as the result of an increasing life span in the general population. Neurodegenerative disorders include a wide range of abnormalities, such as Alzheimer's disease (AD), Parkinson's disease (PD), Creutzfeldt-Jakob disease (CJD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and various neurodegenerative dementias. These diseases generally begin late in life and slowly but inexorably cause progressive neuronal degeneration and result in disability or death. Their diagnosis is difficult for several reasons. These include:

1. The progression of the disease is insidious and the symptoms appear at an advanced stage of the neurodegenerative process (DeKosky and Marek 2003). The diagnosis is then made when the brain lesions are fully constituted.

2. Almost no biological marker is currently available for the routine diagnosis of these abnormalities. Diagnosis relies mainly on clinical examination and neuroimaging techniques (DeKosky and Marek 2003).

3. Differential diagnosis is difficult since there is a considerable overlap between the clinicopathological features of many disorders (Armstrong et al. 2005).

4. The definite diagnosis of many of these conditions, notably CJD, is possible only by post-mortem examination of brain tissue.

The role that proteomics can play in the field of research on neurodegenerative disorders is well described in the goals of the Human Proteome Brain Project launched by the Human Proteome Organisation - HUPO (Hamacher et al. 2004).

9.5.1 Brain Proteome

The systematic proteomic analysis of human or animal brains is of great relevance to neurological diseases. Such studies provide a detailed understanding of the normal and healthy state, and allow comparisons with affected patients or animal models of disease. The first published proteomic studies used 2-DE to produce reference maps of human, mouse and rat brain proteomes. For example, more than 400 proteins were identified from mouse brain (Klose et al. 2002) and more than 300 proteins were identified from human brain (Lubec et al. 2003). The proteins identified corresponded to various functional categories: structural proteins, energy metabolism, protein synthesis, protein degradation, RNA transcription, chaperones, oxidative stress response, signal transduction and synaptic proteins. However, it is likely that the 2-DE analysis of total extracts from whole brain or brain compartments led mainly to the detection of high-abundance proteins. Furthermore, the proteins identified were derived from various cell types: neurons, astrocytes, oligodendrocytes, microglia, blood vessel walls and blood cells.

In recent publications, gel-free technologies were reported for the profiling of the brain proteome. Moreover, efforts were focused on particular subfractions, enabling deeper and more specialised analysis of the proteome. Nielsen et al. (2005) analysed brain plasma membrane proteins using an original fractionation protocol. This procedure was based on high-speed shearing of tissues in solution for removal of soluble proteins, followed by density-gradient enrichment of membrane fractions. Proteins were then digested on-membrane with endoproteinase Lys-C. The resulting peptides were fractionated by reversed-phase chromatography, digested by trypsin and analysed by liquid chromatography (LC)- tandem MS (MS/MS). One advantage of this method is that it requires much lower amounts of tissue than classic membrane-enrichment approaches based on subcellular fractionation. It is thus applicable to small brain compartments and to clinical samples. Use of this method allowed 862 proteins to be identified from 150 mg of mouse brain cortex. After further development and miniaturisation, the authors identified 1,685 proteins from 15 mg of mouse hippocampus. Of the proteins with assigned subcellular localisation, more than 60% were annotated as membrane proteins, including several classes of ion channels and neurotransmitter receptors. In another study, synaptosomal proteins were investigated using ICAT labelling and LC-MS/MS (Schrimpf et al. 2005). Synaptosomes are a subcellular fraction of brain tissue corresponding to isolated synapses. They contain the complete presynaptic terminal and portions of the postsynaptic side. A total of 1,131 proteins were identified, including synaptic adhesion molecules, postsynaptic scaffolding proteins, postsynaptic receptors, postsynaptic receptor-ligands, and proteins involved in synaptic vesicle trafficking or signal transduction cascades.

9.5.2 Proteomic Profiling of Neurodegenerative Disorders

The goal of proteomic studies investigating neurological disorders is to detect differences in protein expression and in protein post-translational modifications that are associated with the disease. The identification of these changes can yield insights into potential molecular mechanisms of neurodegeneration and lead to the identification of potential diagnostic markers or therapeutic targets. For example, the detection of oxidised proteins is of particular interest since oxidative damage is thought to play an important role in the pathogenesis and progression of most, if not all, neurodegenerative disorders.

AD is the major dementia affecting the elderly; thus, it is logical to find a large number of proteomic studies devoted to the investigation of this disease (Butterfield et al. 2003). Proteomic analyses of different brain regions from AD patients and non-demented controls led to the identification of various proteins with altered expression levels. These changes in protein expression reflect the cascade of alterations on multiple pathways within the brain of AD patients: decreased energy metabolism, increased oxidative stress, dysregula-tion of apoptosis, protein misfolding, neurotransmitter imbalance and decreased levels of proteins involved in neuronal cell proliferation, neurite outgrowth or synaptic plasticity. Furthermore, numerous proteins were identified as targets of protein oxidation, which shows the importance of oxidative damage in the progression of the disease (Butterfield et al. 2003). As observed for proteins with altered expression levels, oxidised proteins belong to various functional classes such as energy metabolism, the ubiquitin-proteasome system or neuronal development.

One hallmark of neurodegenerative disorders is the presence of protein deposits in the nervous system. These exist either in the form of extracellular plaques - amyloid plaques in AD or prion protein deposits in CJD - or in the form of intracellular filamentous inclusions - neurofibrillary tangles in AD or Lewy bodies in PD) (Armstrong et al. 2005). Recently, two papers describing the proteomic profiling of such structures after isolation by laser-capture microdissection were published. In the first study, 488 proteins were identified by LC-MS/MS in amyloid plaques isolated from post-mortem AD brain tissues (Liao et al. 2004). Moreover, 26 proteins were found enriched at least twofold in the plaques by quantitative comparison with the adjacent non-plaque regions of the cortex. This approach allowed the discovery of dynein heavy chain in amyloid plaques, a protein involved in intracellular motility of vesicles and organelles along microtubules. The colocalisation of dynein heavy chain with amyloid plaques was corroborated by immunofluorescence confocal microscopy in human AD cortex and in a transgenic mouse model of AD. In the second study, a similar approach was used for the proteomic profiling of neurofibrillary tangles isolated from pyramidal neurons of AD patients (Wang et al. 2005). A total of 155 proteins were tentatively identified, although the majority of these with only one peptide. Of the 72 proteins identified with multiple unique peptides, 63 were not known to be associated with neurofibrillary tangles. Immunohistochemistry experiments confirmed the colocalisation of one of these proteins, glyceraldehyde-3-phosphate dehydro-genase, with neurofibrillary tangles. Further investigations also showed that this protein immunoprecipitates with phosphorylated tau, the major protein component of neurofibrillary tangles, and that it is one of the few proteins known to undergo conversion to a detergent-insoluble form in AD.

Several transgenic animal and cell line models have been developed for the study of neurodegenerative disorders such as AD, PD, CJD, ALS and HD. The R6/2 transgenic mouse model of HD was used in two proteomic studies to identify differentially expressed proteins and oxidative modifications associated with the disease. HD is a hereditary disease caused by a well-characterised genetic defect: the expansion of CAG repeats in exon 1 of the Huntingtin gene. In contrast, the mechanisms by which the mutation causes the disease are not fully understood. The 2-DE analysis of striatum from R6/2 mice and age-matched controls showed that the expression of a-enolase was increased in HD mice (Perluigi et al. 2005). Furthermore, the protein levels of succinyl S-transferase and aspartate aminotransferase were found to increase over the course of the disease, while expression of pyruvate dehydrogenase decreased. In addition, measurement of carbonyl levels led to the detection of six proteins that were oxidised in old versus young R6/2 mice: a-enolase, g-enolase, aconitase, VDAC1, Hsp90 and creatine kinase. Another 2-DE study on R6/2 mice identified two other proteins whose expression decreased in the brain over the course of the disease: a1-antitrypsin and the chaperone aB-crys-tallin (Zabel et al. 2002). Disease progression was also found to be associated with reduced a1-antitrypsin levels in liver and testes, indicating that the disease also exerts its influence outside the brain.

Proteomic techniques were also used to investigate a cell line model of a subset of familial ALS, linked to mutations in the Cu-Zn superoxide dismu-tase SOD1 (Fukada et al. 2004). ALS is a fatal neurodegenerative disorder characterised by progressive motor neuron death. One hypothesis regarding the consequence of SOD1 mutations is the dysregulation of mitochondrial functions and the activation of apoptosis. To explore this hypothesis, mito-chondrial proteins from wild-type NSC34 motor-neuron-like cell lines or those expressing the G93A-SOD1 ALS-causing mutation were analysed by 2-DE. Forty proteins were found to have altered expression in the mutant cell line, including chaperones, subunits of the mitochondrial respiratory chain complexes and two mitochondrial outer-membrane proteins, VDAC1 and VDAC2, both potentially involved in apoptosis.

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