[Show abstract][Hide abstract] ABSTRACT: Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular diseases are known to manifest different clinical symptoms in men and women. Basically this is due to gender-specific genotypes and sexual hormones. We studied gender specificity on the protein expression level in the mouse and human heart, with particular emphasis on the age-dependency of sex-specific protein expression. We first studied the heart proteome in female and male mice at 14 and 100 weeks of age using two-dimensional electrophoresis and mass spectrometry. Protein pattern comparison in young and old mice revealed 7 and 22 protein spots with sex-related expression profiles, respectively. Four proteins co-changed in both age groups. The variant protein spots were identified and revealed 10 distinct proteins and several isoforms thereof: alpha1-antitrypsin (3 isoforms), apolipoprotein A2 (2 isoforms), apolipoprotein A4 (3 isoforms), apolipoprotein E, apolipoprotein J (3 isoforms), carbonic anhydrase 2 (6 isoforms), desmin, nitrilase 1, peroxiredoxin 2 and Rho GDP dissociation inhibitor alpha (2 isoforms). More sex-related proteins were detected in old than in young mice. Through 2DE protein pattern and immunoblot comparisons, six of the variant proteins detected in mice were also observed to change in an age- and sex-dependent manner in the human heart. The age and/or gender-related proteins and species differences in this regard are discussed in terms of cardiovascular disease.
International Journal of Molecular Medicine 01/2008; 20(6):865-74. DOI:10.3892/ijmm.20.6.865 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurodegenerative disorders (ND) encompass clinically and genetically heterogeneous diseases with considerable overlap of their clinical, neuropathological and molecular phenotype. Various causes of neurodegeneration in disease may affect eventually the same proteins within protein networks. To identify common changes in ND, we compared brain protein changes detected by 2-D electrophoresis in four mouse models for ND: (i) Parkinson's disease, (ii) Huntington's disease, (iii) prion disease Scrapie, and (iv) a model for impaired synaptic transmission. To determine specificity of these changes for ND, we extended the scope of our investigation to three neurological conditions that do not result in neurodegeneration (non-ND). We detected 12 to 216 consistent qualitative or quantitative protein changes in individual ND and non-ND models when compared to controls. Up to 36% of these proteins were found to be altered in multiple disease states (at least three) and were therefore termed nodal point proteins. Alterations in alpha B-Crystallin and splicing factor 3b (subunit 4) occurred in at least three ND but not in non-ND. In contrast, alterations in peroxiredoxin 1 and 3, astrocytic phosphoprotein PEA15, complexin 2 and aminoacylase 1 were common to both ND and non-ND. Finally, we investigated the expression pattern of the nodal point proteins in three inbred mouse strains and found different protein abundance (expression polymorphisms) in all cases. Nodal point proteins showing expression polymorphisms may be candidate proteins for disease associated modifiers.
Journal of Proteome Research 09/2006; 5(8):1948-58. DOI:10.1021/pr0601077 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.
[Show abstract][Hide abstract] ABSTRACT: The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.
Cell Death and Differentiation 08/2006; 13(7):1097-109. DOI:10.1038/sj.cdd.4401796 · 8.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The accumulation of mitochondria containing mutated genomes was proposed to be an important factor involved in aging. Although the level of mutated mtDNA has shown to increase over time, it is currently not possible to directly measure the mtDNA mutation rate within living cells. The combination of mathematical modeling and controlled experiments is an alternative approach to obtain an estimate for the mutation rate in a well-defined system. In order to judge the relevance of mitochondrial mutations for the aging process, we used a mouse model to study age-related alterations of the mitochondrial proteins. Based on these experimental data we constructed a mathematical model of the mitochondrial population dynamics to estimate mtDNA mutation rates. Mitochondria were isolated from mouse brain and liver at six different ages (newborn to 24-months). A large-gel 2D-electrophoresis-based proteomics approach was used to analyze the mitochondrial proteins. The expression of two respiratory chain complex I subunits and one complex IV subunit decreased significantly with age. One subunit of complex III and one subunit of complex V increased in expression during aging. Together, these data indicate that complex I and IV deficiency in aged tissues might be accompanied by feedback regulation of other protein complexes in the respiratory chain. When we fitted our experimental data to the mathematical model, mtDNA mutation rate was estimated to be 2.7x10(-8) per mtDNA per day for brain and 3.2x10(-9) per mtDNA per day for liver. According to our model and in agreement with the mitochondrial theory of aging, mtDNA mutations could cause the detrimental changes seen in mitochondrial populations during the normal lifespan of mice, while at the same time ensure that the mitochondrial population remains functional during the developmental and reproductive period of mice.
[Show abstract][Hide abstract] ABSTRACT: Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.