Proteome changes in autosomal recessive primary microcephaly.

BACKGROUND/AIM: Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. MATERIAL AND METHODS: We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. RESULT: We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. CONCLUSION: Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3.

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

MALDI imaging mass spectrometry: discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures.

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin-fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using "bottom-up" MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle.

Comparative proteomic analysis reveals sex and estrogen receptor ß effects in the pressure overloaded heart.

In pressure overload (PO), sex differences in humans and rodents have been well documented and estrogen receptor (ER) ß is considered cardioprotective. However, the underlying mechanisms are poorly understood. Our aim was to investigate sex- and ERß-specific effects in protein abundance in PO employing a 2-dimensional gel electrophoresis/mass spectrometry-based proteomics approach. We hypothesized major sex differences and ERß-specific alterations consistent with cardioprotection in females. Two-month old male and female wild-type (WT) and ERß knockout (BERKO) mice were subjected to transverse aortic constriction (TAC) for 9 weeks (n = 4/group). In WT mice, hypertrophy was significantly more pronounced in males than females, while this sex difference was abolished in BERKO mice. We found 82 protein spots modulated between TAC and sham in WT males, 31 in WT females, 114 in BERKO males, and 87 in BERKO females (P ≤ 0.05). Our analysis revealed in WT and BERKO females an altered pattern of various proteins involved in structure and suggests a link between female sex and cytoskeletal integrity. In males, a set of proteins was identified that associate with mitochondrial bioenergetics and energy supply. We confirmed protein regulation by immunoblotting analysis. In conclusion, the proteomic response of the heart to PO is significantly modulated by ERß and sex. We put forward that the observed differences may identify sex-specific targets for the treatment of heart failure, contributing toward more personalized medical care.

Quantitative and qualitative 2D electrophoretic analysis of differentially expressed mitochondrial proteins from five mouse organs.

Mitochondria fulfill many tissue-specific functions in cell metabolism. We set out to identify differences in the protein composition of mitochondria from five tissues frequently affected by mitochondrial disorders. The proteome of highly purified mitochondria from five mouse organs was separated by high-resolution 2DE. Tissue-specific spots were identified through nano-LC/ESI-MS/MS and quantified by densitometry in ten biological replicates. We identified 87 consistently deviating spots representing 48 proteins. The percentage of variant spots ranged between 4.2% and 6.0%; 21 proteins having tissue-specific isospots. Consistent tissue-specific processing/regulation was seen for carbamoyl-phosphate-synthase, aldehyde-dehydrogenase 2, ATP-synthase α-chain, and isocitrate-dehydrogenase α-subunit. Thirty tissue-specific proteins were associated with mitochondrial disorders in humans. We further identified alcohol-dehydrogenase, catalase, quinone-oxidoreductase, cyclophilin-A, and Upf0317, a potential biotin-carboxyl-carrier protein, which had not been annotated as "mitochondrial" in Gene Ontology or MitoCarta databases. Their targeting to the mitochondria was verified by transfection of full-length GFP-tagged plasmids. Given the high evolutionary conservation of mitochondrial metabolic pathways, these data further annotate the mitochondrial proteome and advance our understanding of the pathophysiology and tissue-specificity of symptoms seen in patients with mitochondrial disorders. The generation of 2D electrophoretic maps of the mitochondrial proteome using tissue specimens in the milligram range facilitates this technique for clinical applications and biomarker research.

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.

Presymptomatic alterations in energy metabolism and oxidative stress in the APP23 mouse model of Alzheimer disease.

Glucose hypometabolism is the earliest symptom observed in the brains of Alzheimer disease (AD) patients. In a former study, we analyzed the cortical proteome of the APP23 mouse model of AD at presymptomatic age (1 month) using a 2-D electrophoresis-based approach. Interestingly, long before amyloidosis can be observed in APP23 mice, proteins associated with energy metabolism were predominantly altered in transgenic as compared to wild-type mice indicating presymptomatic changes in energy metabolism. In the study presented here, we analyzed whether the observed changes were associated with oxidative stress and confirmed our previous findings in primary cortical neurons, which exhibited altered ADP/ATP levels if transgenic APP was expressed. Reactive oxygen species produced during energy metabolism have important roles in cell signaling and homeostasis as they modify proteins. We observed an overall up-regulation of protein oxidation status as shown by increased protein carbonylation in the cortex of presymptomatic APP23 mice. Interestingly, many carbonylated proteins, such as Vilip1 and Syntaxin were associated to synaptic plasticity. This demonstrates an important link between energy metabolism and synaptic function, which is altered in AD. In summary, we demonstrate that changes in cortical energy metabolism and increased protein oxidation precede the amyloidogenic phenotype in a mouse model for AD. These changes might contribute to synaptic failure observed in later disease stages, as synaptic transmission is particularly dependent on energy metabolism.

Mutant Hoxd13 induces extra digits in a mouse model of synpolydactyly directly and by decreasing retinoic acid synthesis.

Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels.

Mesenchymal stromal cells rescue cortical neurons from apoptotic cell death in an in vitro model of cerebral ischemia.

Cell therapy with mesenchymal stromal cells (MSCs) was found to protect neurons from damage after experimental stroke and is currently under investigation in clinical stroke trials. In order to elucidate the mechanisms of MSC-induced neuroprotection, we used the in vitro oxygen­glucose deprivation (OGD) model of cerebral ischemia. Co-culture of primary cortical neurons with MSCs in a transwell co-culture system for 48 h prior to OGD-reduced neuronal cell death by 30-35%. Similar protection from apoptosis was observed with MSC-conditioned media when added 48 h or 30 min prior to OGD, or even after OGD. Western blot analysis revealed increased phosphorylation of STAT3 and Akt in neuronal cultures after treatment with MSC-conditioned media. Inhibition of the PI3K/Akt pathway completely abolished the neuroprotective potential of MSC-conditioned media, suggesting that MSCs can improve neuronal survival by an Akt-dependent anti-apoptotic signaling cascade. Using mass spectrometry, we identified plasminogen activator inhibitor-1 as an active compound in MSC-conditioned media. Thus, paracrine factors secreted by MSCs protect neurons from apoptotic cell death in the OGD model of cerebral ischemia.

Kainate promotes alterations in neuronal RNA splicing machinery.

Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling.

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.

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.

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.

Brief alteration of NMDA or GABAA receptor-mediated neurotransmission has long term effects on the developing cerebral cortex.

Neurotransmitter signaling is essential for physiologic brain development. Sedative and anticonvulsant agents that reduce neuronal excitability via antagonism at N-methyl-D-aspartate receptors (NMDARs) and/or agonism at gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) are applied frequently in obstetric and pediatric medicine. We demonstrated that a 1-day treatment of infant mice at postnatal day 6 (P6) with the NMDAR antagonist dizocilpine or the GABA(A)R agonist phenobarbital not only has acute but also long term effects on the cerebral cortex. Changes of the cerebral cortex proteome 1 day (P7), 1 week (P14), and 4 weeks (P35) following treatment at P6 suggest that a suppression of synaptic neurotransmission during brain development dysregulates proteins associated with apoptosis, oxidative stress, inflammation, cell proliferation, and neuronal circuit formation. These effects appear to be age-dependent as most protein changes did not occur in mice subjected to such pharmacological treatment in adulthood. Previously performed histological evaluations of the brains revealed widespread apoptosis and decreased cell proliferation following such a drug treatment in infancy and are thus consistent with brain protein changes reported in this study. Our results point toward several pathways modulated by a reduction of neuronal excitability that might interfere with critical developmental events and thus affirm concerns about the impact of NMDAR- and/or GABA(A)R-modulating drugs on human brain development.

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.

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

OBJECTIVE: 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. METHODS: Six-day-old C57Bl/6 mice or Wistar rats were exposed to hyperoxia (80% O(2)) or normoxia for 24 hours and received rEpo or normal saline injections intraperitoneally. The amount of degenerating cells in their brains was determined by DeOlmos cupric silver staining. Changes of their brain proteome were determined through two-dimensional electrophoresis and mass spectrometry. Western blot, enzyme activity assays and real-time polymerase chain reaction were performed for further analysis of candidate proteins. RESULTS: Systemic treatment with 20,000 IE/kg rEpo significantly reduced hyperoxia-induced apoptosis and caspase-2, -3, and -8 activity in the brains of infant rodents. In parallel, rEpo inhibited most brain proteome changes observed in infant mice when hyperoxia was applied exclusively. Furthermore, brain proteome changes after a single systemic rEpo treatment point toward a number of mechanisms through which rEpo may generate its protective effect against oxygen toxicity. These include reduction of oxidative stress and restoration of hyperoxia-induced increased levels of proapoptotic factors, as well as decreased levels of neurotrophins. INTERPRETATION: These findings are highly relevant from a clinical perspective because oxygen administration to neonates is often inevitable, and rEpo may serve as an adjunctive neuroprotective therapy.

From 2-D electrophoresis to proteomics.

At first, a short history of the beginning of 2-DE is provided. Based on the present state of the art at the time I developed a 2-DE technique in 1975 that was able to resolve complex protein extracts from mouse tissues in hundreds of protein spots. My intention was to study proteins from a global point of view. Questions of interest were, how do proteins change during embryonic development, and what is the effect of induced mutations on the protein level. At that time protein chemistry was a matter of analyzing single proteins in detail. Therefore, my approach was frequently criticized as inappropriate because it would be impossible to identify and characterize the hundreds of proteins resolved. But soon it was realized that studying total proteins gives opportunities to answer many interesting questions. This led to a research field nowadays called "proteomics". Already in the beginning of the 1980s the idea to analyze the total human proteins had come up. By entering the post-genome era it became obvious that a human proteome project is needed in order to explain the human genome in terms of its functions. The problems in realizing such a project are considered.

Protein extraction for 2DE.

Our protein extraction protocol for two-dimensional gel electrophoresis (2DE) was updated to meet current needs in the field of proteomics. This protocol summarizes our experience using this method since its introduction over 30 years ago. We provide a total as well as fractionated extraction protocol. The former is easy and fast to use, suitable for most standard 2DE applications, whereas the latter is used for special applications such as the extraction of membrane or nuclear proteins.Both extraction protocols stress the need that protease inhibitors are added early to still deep frozen tissue to preclude an activation of proteases which destroy proteins and make them inaccessible to analysis. We also emphasize that, to remain soluble, proteins need to stay in an environment resembling a living cell as closely as possible. Sample dilution is therefore kept to a minimum and the pH of the extract is close to in vivo conditions at pH 7.1. In addition there are no precipitation/resolubilization steps which could irreversibly remove proteins from the extract. Furthermore, the total extraction does not even require centrifugation. Our extraction protocol is compatible with recent advances in 2DE-staining techniques such as differential in gel electrophoresis and fluorescence staining as well as mass spectrometry.

High-resolution large-gel 2DE.

Our two-dimensional gel electrophoresis (2DE) protocol has been continuously improved in our laboratory since its inception 30 years ago. An updated version is presented here. This protocol is a result of our experience in proteome analysis of tissue extracts, cultured cells (mammalian, yeast, and bacteria), cellular organelles, and subcellular fractions. Many modifications and suggestions emerging in our lab as well as in the literature were tested and integrated into our improved protocol if helpful. Importantly we use (a) large (46 x 30 cm) gels to achieve a high resolution and (b) ready-made gel solutions produced in large batches and stored frozen, a prerequisite, among others, for our very high reproducibility. Employing the 2DE method described here we demonstrated that protein patterns separating more than 10,000 protein spots can be obtained from mouse tissue. This is the highest resolution reported in the literature for 2DE of complex protein mixtures so far. Our 2DE patterns are of high quality with regard to spot shape and intensity as well as background. The reproducibility of the protein patterns is shown to be extremely satisfactory. New staining methods such as differential in gel electrophoresis (DIGE) and the latest 2DE gel evaluation software are compatible to our 2DE protocol. Using suitable staining protocols proteins can easily be identified by mass spectrometry.