SWATH analysis of the synaptic proteome in Alzheimer's disease.

Brain tissue from Alzheimer's disease patients exhibits synaptic degeneration in selected regions. Synaptic dysfunction occurs early in the disease and is a primary pathological target for treatment. The molecular mechanisms underlying this degeneration remain unknown. Quantifying the synaptic proteome in autopsy brain and comparing tissue from Alzheimer's disease cases and subjects with normal aging are critical to understanding the molecular mechanisms associated with Alzheimer pathology. We isolated synaptosomes from hippocampus and motor cortex so as to reduce sample complexity relative to whole-tissue homogenates. Synaptosomal extracts were subjected to strong cation exchange (SCX) fractionation to further partition sample complexity; each fraction received SWATH-based information-dependent acquisition to generate a comprehensive peptide-ion library. The expression of synaptic proteins from AD hippocampus and motor cortex was then compared between groups. A total of 2077 unique proteins were identified at a critical local false discovery rate <5%. Thirty of these, including 17 novel proteins, exhibited significant expression differences between cases and controls; these proteins are involved in cellular functions including structural maintenance, signal transduction, autophagy, oxidative stress, and proteasome activity, or they have synaptic-vesicle related or energy-related functions. Differentially expressed proteins were subjected to pathway analysis to identify protein-protein interactions. This revealed that the most perturbed molecular and cellular functions were cellular assembly and organization. Core analysis revealed RhoA signaling to be the top canonical pathway. Network analysis showed that differentially expressed proteins were related to cellular assembly and organization, and cellular function and maintenance. This is the first study to combine SCX fractionation with SWATH analysis. SWATH is a promising new technique that can greatly enhance protein identification in any proteome, and has many other benefits; however, there are limitations yet to be resolved.

Expression of CYP2E1 and CYP2U1 proteins in amygdala and prefrontal cortex: influence of alcoholism and smoking.

BACKGROUND: The tissue-specific expression of cytochrome P450 enzymes (CYP, P450) in the human brain may influence the therapeutic response to, and side effects of, neuroactive drugs including alcohol. However, the distribution of many P450s, especially poorly characterized CYP2 forms, within specific regions of the brain remains obscure, partly due to the paucity of available tissue and difficulty in discriminating between related P450s with available antibodies. METHODS: In this study, we analyzed the expression of CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, and CYP2W1 proteins in human prefrontal cortex (PFC) and amygdala (AMG) by immunoblotting with antibodies for which the P450 form specificity had been enhanced by affinity purification. These brain regions were selected as they mediate the addictive effects of cigarette smoking and alcohol consumption, substances known to modulate P450 expression in other tissues. PFC and AMG samples from alcoholic smokers, alcoholic nonsmokers, nonalcoholic smokers, and nonalcoholic nonsmokers were studied to assess the effect of alcohol use and smoking on the expression of these proteins. RESULTS: Of the P450s studied, CYP2E1 and CYP2U1 were expressed in all samples analyzed (n = 26 and 22 for CYP2E1 and CYP2U1, respectively), and elevated in alcoholics. CYP2U1 expression was also slightly increased in smokers. Expression of both P450s was increased in AMG compared to PFC of the same individuals. CONCLUSIONS: This is the first report of CYP2E1 and CYP2U1 protein expression in human AMG. Our results suggest that CYP2U1 expression may be modulated by alcohol and tobacco, with potential consequent effects on the metabolism of drugs and endogenous chemicals by this enzyme.

Targeted quantitative analysis of synaptic proteins in Alzheimer's disease brain.

Brain tissue from Alzheimer's disease (AD) patients shows significant loss of synapses in selected regions. Synaptic degeneration is the best predictor for loss of cognitive functions ante mortem. The molecular mechanisms underlying this degeneration remain unknown. Our previous two-dimensional gel-electrophoresis proteomics study found that 26 synaptic proteins are differentially expressed in Alzheimer's brain. It is difficult to quantify global protein expression using this technique because (a) several proteins can migrate together and (b) isoforms of the same protein can migrate to different places. The present study estimated global synaptic protein levels by label-free multiple reaction monitoring. Multiple reaction monitoring is a powerful and sensitive mass spectrometry technique that specifically targets multiple protein of interests. The severely AD-affected hippocampus was compared with motor cortex, a relatively spared region. We targeted ten proteins in autopsy brain based on the earlier study. Analytes separated by high performance liquid-chromatography were monitored on a hybrid triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. With the use of an internal standard protein, linear and highly reproducible (CV<9%) label-free assays were achieved. Data were contrasted with the gel-based study to highlight differences and similarities. Significantly higher expression levels of peroxiredoxin-1 (may provide antioxidant protection) and dihydropyrimidinase-related protein-1 (associated with cytoskeletal remodeling) were found in AD hippocampus. Significantly lower levels of peroxiredoxin-1 and the energy-related enzymes creatine kinase B and fructose-bisphosphate aldolase C were found in non-AD hippocampus. Our previously reported difference in synaptotagmin expression is probably isoform-specific. These findings suggest potential roles of key proteins in synaptic loss in AD, and/or a protective mechanism in non-AD brain tissue.

Quantitative multiple reaction monitoring analysis of synaptic proteins from human brain.

BACKGROUND: The recent introduction of multiple reaction monitoring to proteomics research has allowed many researchers to apply this technique to study human diseases. NEW METHODS: Here we combine subcellular fractionation of human autopsy brain with label-free multiple reaction monitoring to quantitatively analyse proteins in synapses. The protein enolase, from Streptococcus pyogenes serotype M6, which is sufficiently different from human proteins, was spiked into the sample mixture prior to trypsin digestion and used as an internal standard across samples. RESULTS: Three synaptic proteins and an internal standard analysed with four injections over four consecutive days gave consistent differences with a coefficient of variation of <4%. Consistent retention time was recorded across the replicates. Comparison with existing methods: Previously, multiple reaction monitoring analysis has been utilized to study human autopsy and animal tissues. Utilizing the synaptosomal fraction prior to analysis reduced sample complexity and allowed the enriched synaptic proteins to be quantitatively assessed in a highly reproducible manner, without the need for expensive fluorescent labels and synthetic peptides. CONCLUSION: Protein expression can be measured with accuracy using label-free multiple reaction monitoring mass spectrometry in relatively complex human brain samples. Synaptic functions are critical for neuronal communication and function, and synapse dysfunction underlies many neurodegenerative diseases, including Alzheimer's disease. This method can be applied to study a range of brain disorders.

The synaptic proteome in Alzheimer's disease.

BACKGROUND: Synaptic dysfunction occurs early in Alzheimer's disease (AD) and is recognized to be a primary pathological target for treatment. Synapse degeneration or dysfunction contributes to clinical signs of dementia through altered neuronal communication; the degree of synaptic loss correlates strongly with cognitive impairment. The molecular mechanisms underlying synaptic degeneration are still unclear, and identifying abnormally expressed synaptic proteins in AD brain will help to elucidate such mechanisms and to identify therapeutic targets that might slow AD progression. METHODS: Synaptosomal fractions from human autopsy brain tissue from subjects with AD (n = 6) and without AD (n = 6) were compared using two-dimensional differential in-gel electrophoresis. AD pathology is region specific; human subjects can be highly variable in age, medication, and other factors. To counter these factors, two vulnerable areas (the hippocampus and the temporal cortex) were compared with two relatively spared areas (the motor and occipital cortices) within each group. Proteins exhibiting significant changes in expression were identified (≥20% change, Newman-Keuls P value < .05) using either matrix-assisted laser desorption ionization time-of-flight or electrospray ionisation quadrupole-time of flight mass spectrometry. RESULTS: Twenty-six different synaptic proteins exhibited more than twofold differences in expression between AD and normal subjects. These proteins are involved in regulating different cellular functions, including energy metabolism, signal transduction, vesicle transport, structure, and antioxidant activity. CONCLUSION: Comparative proteome analysis uncovered markers of pathogenic mechanisms involved in synaptic dysfunction.

Synaptic proteome changes in the superior frontal gyrus and occipital cortex of the alcoholic brain.

Cognitive deficits and behavioral changes that result from chronic alcohol abuse are a consequence of neuropathological changes which alter signal transmission through the neural network. To focus on the changes that occur at the point of connection between the neural network cells, synaptosomal preparations from post-mortem human brain of six chronic alcoholics and six non-alcoholic controls were compared using 2D-DIGE. Functionally affected and spared regions (superior frontal gyrus, SFG, and occipital cortex, OC, respectively) were analyzed from both groups to further investigate the specific pathological response that alcoholism has on the brain. Forty-nine proteins were differentially regulated between the SFG of alcoholics and the SFG of controls and 94 proteins were regulated in the OC with an overlap of 23 proteins. Additionally, the SFG was compared to the OC within each group (alcoholics or controls) to identify region specific differences. A selection were identified by MALDI-TOF mass spectrometry revealing proteins involved in vesicle transport, metabolism, folding and trafficking, and signal transduction, all of which have the potential to influence synaptic activity. A number of proteins identified in this study have been previously related to alcoholism; however, the focus on synaptic proteins has also uncovered novel alcoholism-affected proteins. Further exploration of these proteins will illuminate the mechanisms altering synaptic plasticity, and thus neuronal signaling and response, in the alcoholic brain.

Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development.

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis.

Protein degradation plays an important role in modulating ethylene signal transduction in plants. Here we show that the ethylene receptor ETR2 is one such target for degradation and that its degradation is dependent upon perception of the signaling ligand ethylene. The ETR2 protein is initially induced by ethylene treatment, consistent with an increase in transcript levels. At ethylene concentrations above 1 mul/liter, however, ETR2 protein levels subsequently decrease in a post-transcriptional fashion. Genetic and chemical approaches indicate that ethylene perception by the receptors initiates the reduction in ETR2 protein levels. The ethylene-induced decrease in ETR2 levels is not affected by cycloheximide, an inhibitor of protein biosynthesis, but is affected by proteasome inhibitors, indicating a role for the proteasome in ETR2 degradation. Ethylene-induced degradation still occurs in seedlings treated with brefeldin A, indicating that degradation of ETR2 does not require exit from its subcellular location at the endoplasmic reticulum. These data support a model in which ETR2 is degraded by a proteasome-dependent pathway in response to ethylene binding. Implications of this model for ethylene signaling are discussed.

Dissecting the ethylene pathway of Arabidopsis.

The plant hormone ethylene regulates growth, development and stress responses. In recent years, various genomic and proteomic approaches have been initiated to understand both the range of ethylene responses in the plant and the mechanism of signal transduction. Transcriptional profiling experiments reveal broad-ranging effects of ethylene upon gene regulation, with up to 7 per cent of the genes examined demonstrating a significant level of response in one study. Both transcriptional and post-transcriptional mechanisms regulate the expression of components within the ethylene signal transduction pathway. The importance of post-transcriptional regulation via the ubiquitin/proteasome-mediated degradation pathway is apparent in studies on the accumulation of ethylene insensitive 3 (EIN3), a key transcription factor in the pathway. Protein complexes also play a role in modulating ethylene signal transduction, with interactions between the ethylene receptors and the Raf-like kinase constitutive triple response-1 (CTR1) being required for ethylene perception at the endoplasmic reticulum. In this paper, recent developments in unravelling the transcriptional and post-transcriptional regulation of the ethylene signalling and response pathways are considered, along with the latest developments in unravelling the biochemical mechanism behind ethylene perception.

Ethylene signal transduction.

BACKGROUND: The phytohormone ethylene is a key regulator of plant growth and development. Components of the pathway for ethylene signal transduction were identified by genetic approaches in Arabidopsis and have now been shown to function in agronomically important plants as well. SCOPE: This review focuses on recent advances in our knowledge on ethylene signal transduction, in particular on recently proposed components of the pathway, on the interaction between the pathway components and on the roles of transcriptional and post-transcriptional regulation in ethylene signalling. CONCLUSIONS: Data indicate that the site of ethylene perception is at the endoplasmic reticulum and point to the importance of protein complexes in mediating the initial steps in ethylene signal transduction. The expression level of pathway components is regulated by both transcriptional and post-transcriptional mechanisms, degradation of the transcription factor EIN3 being a primary means by which the sensitivity of plants to ethylene is regulated. EIN3 also represents a control point for cross-talk with other signalling pathways, as exemplified by the effects of glucose upon its expression level. Amplification of the initial ethylene signal is likely to play a significant role in signal transduction and several mechanisms exist by which this may occur based on properties of known pathway components. Signal output from the pathway is mediated in part by carefully orchestrated changes in gene expression, the breadth of these changes now becoming clear through expression analysis using microarrays.