Brain region specific mitophagy capacity could contribute to selective neuronal vulnerability in Parkinson's disease.

Parkinson's disease (PD) is histologically well defined by its characteristic degeneration of dopaminergic neurons in the substantia nigra pars compacta. Remarkably, divergent PD-related mutations can generate comparable brain region specific pathologies. This indicates that some intrinsic region-specificity respecting differential neuron vulnerability exists, which codetermines the disease progression. To gain insight into the pathomechanism of PD, we investigated protein expression and protein oxidation patterns of three different brain regions in a PD mouse model, the PINK1 knockout mice (PINK1-KO), in comparison to wild type control mice. The dysfunction of PINK1 presumably affects mitochondrial turnover by disturbing mitochondrial autophagic pathways. The three brain regions investigated are the midbrain, which is the location of substantia nigra; striatum, the major efferent region of substantia nigra; and cerebral cortex, which is more distal to PD pathology. In all three regions, mitochondrial proteins responsible for energy metabolism and membrane potential were significantly altered in the PINK1-KO mice, but with very different region specific accents in terms of up/down-regulations. This suggests that disturbed mitophagy presumably induced by PINK1 knockout has heterogeneous impacts on different brain regions. Specifically, the midbrain tissue seems to be most severely hit by defective mitochondrial turnover, whereas cortex and striatum could compensate for mitophagy nonfunction by feedback stimulation of other catabolic programs. In addition, cerebral cortex tissues showed the mildest level of protein oxidation in both PINK1-KO and wild type mice, indicating either a better oxidative protection or less reactive oxygen species (ROS) pressure in this brain region. Ultra-structural histological examination in normal mouse brain revealed higher incidences of mitophagy vacuoles in cerebral cortex than in striatum and substantia nigra. Taken together, the delicate balance between oxidative protection and mitophagy capacity in different brain regions could contribute to brain region-specific pathological patterns in PD.

Superoxide dismutase type 1 in monocytes of chronic kidney disease patients.

We analyzed proteomic profiles in monocytes of chronic kidney disease (CKD) patients and healthy control subjects. Two-dimensional electrophoresis (2-DE) and silver staining indicated differences in protein pattern. Among the analyzed proteins, superoxide dismutase type 1 (SOD1), which was identified both by MS/MS mass-spectrometry and immunoblotting, was reduced in kidney disease. We characterized SOD1 protein amount, using quantitative in-cell Western assay and immunostaining of 2-DE gel blots, and SOD1 gene expression, using quantitative real-time polymerase chain reaction (PCR), in 98 chronic hemodialysis (HD) and 211 CKD patients, and 34 control subjects. Furthermore, we showed that different SOD1 protein species exist in human monocytes. SOD1 protein amount was significantly lower in HD (normalized SOD1 protein, 27.2 ± 2.8) compared to CKD patients (34.3 ± 2.8), or control subjects (48.0 ± 8.6; mean ± SEM; P < 0.05). Analysis of SOD1 immunostaining showed significantly more SOD1 protein in control subjects compared to patients with CKD or HD (P < 0.0001, analysis of main immunoreactive protein spot). SOD1 gene expression was significantly higher in HD (normalized SOD1 gene expression, 17.8 ± 2.3) compared to CKD patients (9.0 ± 0.7), or control subjects (5.5 ± 1.0; P < 0.0001). An increased SOD1 gene expression may indicate increased protein degradation in patients with CKD and compensatory increase of SOD1 gene expression. Taken together, we show reduced SOD1 protein amount in monocytes of CKD, most pronounced in HD patients, accompanied by increased SOD1 gene expression.

PROTEOMER: A workflow-optimized laboratory information management system for 2-D electrophoresis-centered proteomics.

In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large-scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2-DE/MS-based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2-DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2-DE experiments with MS-data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our "PROTEOMER" database is its high cross-referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross-referencing can transform information into biological knowledge.

Proteome analysis of ventral midbrain in MPTP-treated normal and L1cam transgenic mice.

Treatment of mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridene hydrochloride (MPTP) is a well established animal model for Parkinson's disease (PD), while overexpression of L1 cell adhesion molecule (L1cam) has been proposed to attenuate the degeneration of dopaminergic neurons induced by MPTP. To gain insight into the role of L1cam in the pathomechanism of PD, we investigated protein expression patterns after MPTP-treatment in both C57BL/6 (wild-type) and transgenic mice overexpressing L1cam in astrocytes. Our results showed that during the acute phase, proteins in functional complexes responsible for mitochondrial, glycolysis, and cytoskeletal function were down-regulated in MPTP-treated wild-type mice. After a recovery phase, proteins that were down-regulated in the acute phase reverted to normal levels. In L1cam transgenic mice, a much higher number of proteins was altered during the acute phase and this number even increased after the recovery phase. Many proteins involved in oxidative phosphorylation were still down-regulated and glycolysis related protein were still up-regulated. This pattern indicates a lasting severely impaired energy production in L1cam mice after MPTP treatment.

Heart protein expression related to age and sex in mice and humans.

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.