Joachim Klose

Genetic analysis of the mouse brain proteome

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.

Epigenetic inheritance in the mouse

Acquired epigenetic modifications, such as DNA methylation or stable chromatin structures, are not normally thought to be inherited through the germline to future generations in mammals [1] [2]. Studies in the mouse have shown that specific manipulations of early embryos, such as nuclear transplantation, can result in altered patterns of gene expression and induce phenotypic alterations at later stages of development [3] [4] [5]. These effects are consistent with acquired epigenetic modifications that are somatically heritable, such as DNA methylation. Repression and DNA methylation of genes encoding major urinary proteins, repression of the gene encoding olfactory marker protein, and reduced body weight can be experimentally induced by nuclear transplantation in early embryos [4]. Strikingly, we now report that these acquired phenotypes are transmitted to most of the offspring of manipulated parent mice. This is the first demonstration of epigenetic inheritance of specific alterations of gene expression through the germline. These observations establish a mammalian model for transgenerational effects that are important for humal health, and also raise the question of the evolutionary importance of epigenetic inheritance.

Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague–Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High‐resolution two‐dimensional electrophoresis of the MSC proteome, under conditions of in vitro self‐renewal as well as osteogenic stimulation, identified several age‐dependent proteins, including members of the calponin protein family as well as galectin‐3. Functional annotation clustering revealed that age‐affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC‐based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation. STEM CELLS 2009;27:1288–1297

Analysis of the mouse proteome. (I) Brain proteins: Separation by two‐dimensional electrophoresis and identification by mass spectrometry and genetic variation

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large‐gel two‐dimensional electrophoresis. Two‐dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer‐assisted image analysis and treatment with our spot detection program. The resulting two‐dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix‐assisted laser desorption/ionization ‐ mass spectrometry (MALDI‐MS) after in‐gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two‐dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post‐translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.

Genotypes and phenotypes

Within the framework of a pilot project on the analysis of the mouse proteome, we investigated C57BL/6 mice (Mus musculus), a standard inbred strain of the mouse, starting with the analysis of brain, liver and heart proteins. Tissue extraction and the separation of proteins were performed with techniques offering a maximum of resolution. Proteins separated were analyzed by mass spectrometry. Gene‐protein identification was performed by genetic analyses using the European Collaborative Interspecific Backcross (EUCIB), established from the two mouse species Mus musculus and Mus spretus. On the basis of protein polymorphisms we mapped hundreds of genes on the mouse chromosomes, allowing us new insight into the relationship between genotype and phenotype of proteins. In particular, the results showed that protein modifications can be genetically determined, therefore representing their own class of protein phenotypes. In this context, results are discussed suggesting that phenotypes of single protein species may result from several genes. Accordingly, proteins are considered as polygenic traits. In contrast, one example demonstrates that proteins may also have pleiotropic effects: a single gene mutation (a single altered protein) may affect several other proteins. From these studies we conclude that gene‐related functional proteomics will show in the future that genetic diseases, defined today by clinical symptoms and considered as etiological entireties, can be subdivided into different diseases according to different affected genes.

Alterations in the Mouse and Human Proteome Caused by Huntington’s Disease

Huntington’s disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that α1-antitrypsin and αB-crystalline both decrease in expression over the course of disease. Importantly, the α1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors α1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone αB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington’s disease patients, α1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining α1-antitrypsin and αB-crystallin availability during the course of Huntington’s disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.

A Large Number of Protein Expression Changes Occur Early in Life and Precede Phenotype Onset in a Mouse Model for Huntington Disease

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.

Erythropoietin protects the developing brain from hyperoxia-induced cell death and proteome changes.

Oxygen toxicity has been identified as a risk factor for adverse neurological outcome in survivors of preterm birth. In infant rodent brains, hyperoxia induces disseminated apoptotic neurodegeneration. Because a tissue‐protective effect has been observed for recombinant erythropoietin (rEpo), widely used in neonatal medicine for its hematopoietic effect, we examined the effect of rEpo on hyperoxia‐induced brain damage.

Genetic variability of soluble proteins studied by two-dimensional electrophoresis on different inbred mouse strains and on different mouse organs.

SummaryThe soluble proteins of four organs (liver, kidney, brain and muscle) of mice from four inbred strains (C57BL/6J, DBA/2J, AKR/J and BALB/cHan) and offspring from cross-breedings therefrom are investigated for genetic variants. The female mice from each strain are divided into different groups according to age (12–14 and 24–26 weeks) and generation (P and F1). The proteins are separated by two-dimensional gel electrophoresis (2DE) using two different techniques (2D GE and 2D SDS-GE), developed in our laboratory.