Identification of proteomic changes during differentiation of adult mouse subventricular zone progenitor cells.

The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features that control NPC proliferation and their differentiation into neurons, astrocytes, and oligodendrocytes are unclear. In the present study, we used a comparative proteomics approach to identify proteins that were differentially regulated in NPCs after short-term differentiation. We also used a subcellular fractionation technique for enrichment of nuclei and other dense organelles to identify proteins that were not readily detected in whole cell extracts. In total, 115 distinct proteins underwent expression changes during NPC differentiation. Forty one of these were only identified following subcellular fractionation. These included transcription factors, RNA-processing factors, cell cycle proteins, and proteins that translocate between the nucleus and cytoplasm. Biological network analysis showed that the differentiation of NPCs was associated with significant changes in cell cycle and protein synthesis machinery. Further characterization of these proteins could provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system (CNS) and potentially identify points of therapeutic intervention.

Identification of differentiating neural progenitor cell markers using shotgun isobaric tagging mass spectrometry.

The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. This requires an understanding of the molecular events and biological features that regulate the self-renewal of NPCs and their differentiation into neurons, astrocytes, and oligodendendrocytes. In this study, we have characterized the proteomic changes that occur upon differentiation of these cells using the novel iTRAQ labeling chemistry for quantitative mass spectrometry. In total, 55 distinct proteins underwent expression changes during NPC differentiation. This included 14 proteins that were identified by our previous two-dimensional difference gel electrophoresis (2D-DIGE) analysis of differentiating mouse neurospheres. The importance of the iTRAQ approach was demonstrated by the identification of additional proteins that were not resolved by the 2D-DIGE technology. The proteins identified by the iTRAQ approach included growth factors, signaling molecules, proliferating cell-specific proteins, heat shock proteins, and other proteins involved in the regulation of metabolism and the transcriptional and translational machinery. Further characterization of the identified proteins should provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system and potentially that of other proliferating cell types, including peripheral stem cells or cancer cells.

Proteomic analysis identifies alterations in cellular morphology and cell death pathways in mouse brain after chronic corticosterone treatment.

Some patients with Major Depression and other neurological afflictions display hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. HPA hyperactivity may be due to impaired feedback inhibition and manifested as increased levels of circulating cortisol. Subcutaneous implants of corticosterone pellets were used to mimic this situation in mice to gain insight into any effects on brain function by comparative proteomic analysis using two-dimensional Differential In-Gel Electrophoresis. A total of 150 different protein spots were altered by corticosterone treatment in the hypothalamus, hippocampus and cerebral cortex. Of these, 117 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting equating to 51 different proteins. Association of these corticosterone-modulated proteins with biological functions using the Ingenuity Pathways Analysis tool showed that cell morphology was significantly altered in the hippocampus and cerebral cortex, whereas the hypothalamus showed significant changes in cell death. Ingenuity Pathways Analysis of the canonical signaling pathways showed that glycolysis and gluconeogenesis were altered in the hypothalamus and the hippocampus and all three brain regions showed changes in phenylalanine, glutamate and nitrogen metabolism. Further elucidation of these pathways could lead to identification of biomarkers for the development of pharmacological therapies targeted at neuropsychiatric disorders.

Mechanisms of action of the antidepressants fluoxetine and the substance P antagonist L-000760735 are associated with altered neurofilaments and synaptic remodeling.

Antidepressants are widely prescribed in the treatment of depression, although the mechanism of how they exert their therapeutic effects is poorly understood. To shed further light on their mode of action, we have attempted to identify a common proteomic signature in guinea pig brains after chronic treatment with two different antidepressants. Both fluoxetine and the substance P receptor (NK(1)R) antagonist (SPA) L-000760735 altered cortical expression of multiple heat shock protein 60 forms along with neurofilaments and related proteins that are critical determinants of synaptic structure and function. Analysis of NK(1)R-/- mice showed similar alterations of neurofilaments confirming the specificity of the effects observed with chronic NK(1)R antagonist treatment. To determine if these changes were associated with structural modification of synapses, we carried out electron microscopic analysis of cerebral cortices from fluoxetine-treated guinea pigs. This showed an increase in the percentage of synapses with split postsynaptic densities (PSDs), a phenomenon that is characteristic of activity-dependent synaptic rearrangement. These findings suggest that cortical alterations of the neurofilament pathway and increased synaptic remodeling are associated with the mechanism of these two antidepressant drug treatments and may contribute to their psychotherapeutic actions.

Detection of gender differences in rat lens proteins using 2-D-DIGE.

The glass-like transparency of the human eye lens is achieved by the tight packing of abundant crystallin proteins. However, the precise role of the accessory non-crystallin proteins is not well understood. We have carried out 2-DE mapping of these proteins in rat lens. This showed the presence of the high molecular weight filamentous structural proteins spectrin, filensin, tubulin, vimentin, actin and phakinin as well as several forms of potential crystallin oligomers comprised of alphaA, betaB1, betaA1 and betaA4 chains. Other proteins that were present include, heat shock protein 71, WD repeat protein 1, and several enzymes including alpha-enolase, pyruvate kinase, transketolase and aldose reductase. 2-D-DIGE analysis revealed several expression differences between the lens proteomes of male and female rats. Female rat lenses contained lower levels of aldose reductase, increased proteolyic fragments of the structural proteins filensin, vimentin and phakinin and higher levels of potential alphaA, betaB1 and betaA1 crystallin oligomers. Taken together these findings suggest that there are potential differences in oxidative stress regulation between male and female rat lenses, which may have implications on susceptibility to cataract formation. Future studies aimed at elucidating pre-cataractic changes in the non-crystallin proteins described here may facilitate identification of novel markers involved in cataractogenesis.

Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis.

This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK(1)R-/-) and wild type (NK(1)R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK(1)R-/- and NK(1)R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3- and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK(1)R-/- mice compared to NK(1)R+/+ controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis.

Transgenic, knockout and knockin mice are useful tools for linking specific genes with behaviour and other complex biological processes. However, complications arising due to compensatory changes, genetic background differences and other factors could lead to difficulty in interpreting the resulting changes in phenotype. We have used fluorescence two-dimensional differential in-gel electrophoresis in combination with matrix-assisted laser desorption/ionization-time of flight mass fingerprinting to investigate the possibility that distinct genetic alterations can lead to common protein expression changes in genetically modified mice. Brain proteomes were compared from two transgenic mouse strains (Tg2576 x TgPS1 and Tg2576), two knockout mouse strains (5-HT(7)R -/- and GABA(A)Ralpha5 -/-) and one knockin mouse strain (GABA(A)Ralpha1-H101R). Both of the transgenic models showed an isoform change in the heat shock 70 related protein, mortalin. The knockout and knockin models showed similar changes in mortalin expression along with an alteration of the anti-oxidant protein 2. The observed proteomic alterations indicate that stress-responsive protein pathways may be altered artefactually in all of the mouse models used in this study and highlights an area where caution is needed in interpreting proteomic changes in genetically modified mice.

A proteomic investigation of drug-induced steatosis in rat liver.

A significant problem faced by pharmaceutical companies today is the failure of lead compounds in the later stages of development due to unexpected toxicities. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify a proteomic signature associated with hepatocellular steatosis in rats after dosing with a compound in preclinical development. Liver toxicity was monitored over a 5 day dosing regime using blood biochemical parameter measurements and histopathological analysis. As early as 6 h postdosing, livers showed hepatocellular vacuolation, which increased in extent and severity over the course of the study. Alterations in plasma glucose, alanine aminotransferase, and aspartate aminotransferase were not detected until the third day of dosing and changed in magnitude up to the final day. The proteomic changes were observed at the earliest time point, and many of these could be associated with known toxicological mechanisms involved in liver steatosis. This included up-regulation of pyruvate dehydrogenase, phenylalanine hydroxylase, and 2-oxoisovalerate dehydrogenase, which are involved in acetyl-CoA production, and down-regulation of sulfite oxidase, which could play a role in triglyceride accumulation. In addition, down-regulation of the chaperone-like protein, glucose-regulated protein 78, was consistent with the decreased expression of the secretory proteins serum paraoxonase, serum albumin, and peroxiredoxin IV. The correlation of these protein changes with the clinical and histological data and their occurrence before the onset of the biochemical changes suggest that they could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis.

Oligomerization of G-protein-coupled receptors shown by selective co-immunoprecipitation.

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.