Naomi Etheridge

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 that 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 2‐D differential in‐gel electrophoresis (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 was 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.

SWATH analysis of the synaptic proteome in Alzheimer's disease

Brain tissue from Alzheimers 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 Alzheimers 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.

The synaptic proteome in Alzheimer's disease

Synaptic dysfunction occurs early in Alzheimers 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.

Targeted quantitative analysis of synaptic proteins in Alzheimer’s disease brain

Brain tissue from Alzheimers 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 Alzheimers 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 Alzheimers disease. This method can be applied to study a range of brain disorders.

Identifying changes in the synaptic proteome of cirrhotic alcoholic superior frontal gyrus.

Hepatic complications are a common side-effect of alcoholism. Without the detoxification capabilities of the liver, alcohol misuse induces changes in gene and protein expression throughout the body. A global proteomics approach was used to identify these protein changes in the brain. We utilised human autopsy tissue from the superior frontal gyrus (SFG) of six cirrhotic alcoholics, six alcoholics without comorbid disease, and six non-alcoholic non-cirrhotic controls. Synaptic proteins were isolated and used in two-dimensional differential in-gel electrophoresis coupled with mass spectrometry. Many expression differences were confined to one or other alcoholic sub-group. Cirrhotic alcoholics showed 99 differences in protein expression levels from controls, of which half also differed from non-comorbid alcoholics. This may reflect differences in disease severity between the sub-groups of alcoholics, or differences in patterns of harmful drinking. Alternatively, the protein profiles may result from differences between cirrhotic and non-comorbid alcoholics in subjects’ responses to alcohol misuse. Ten proteins were identified in at least two spots on the 2D gel; they were involved in basal energy metabolism, synaptic vesicle recycling, and chaperoning. These post-translationally modified isoforms were differentially regulated in cirrhotic alcoholics, indicating a level of epigenetic control not previously observed in this disorder.

Effects of alcohol on the brain

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Identifying Changes in the alcoholic synaptic proteome

MicroRNAs (miRNAs) represent a large class of small single-stranded non-coding RNAs that are thought to regulate diverse functions by post-transcriptional gene silencing. Recently, miRNAs are have been shown to be crucial regulators of gene expression, affecting a wide variety of cellular functions. In brain, miRNAs have been shown to play an important role during development and in regulation of synaptic plasticity, and have been implicated in human brain diseases. In previous studies, we and others have used cDNA and oligonucleotide microarrays to identify and classify genes with altered expression following long-term alcohol consumption; however, the potential role of miRNA regulation of these genes has not been investigated. In the present study, we used miRNA arrays (Ambion+Invitrogen human, mouse, and rat probes) to classify miRNAs with altered expression in postmortem prefrontal cortex following long-term alcohol consumption. miRNAs profiles were obtained from the identical samples (14 well characterized alcoholics and 13 matched controls) that were used in a previous cDNA microarray study (Neuropsychopharm 31, 1574-82, 2006). We found that ~20 miRNAs were significantly up-regulated (p<0.002; 20-45%) in the alcoholic group compared to controls. Interestingly, miRNA down-regulation was not observed at this level of significance. Functional classification of the predicted target genes of the regulated miRNAs demonstrated a large degree of overlap with published mRNA data. For example, genes involved in ubiquitination, apoptosis, cell adhesion, neurogenesis, and neural disease are predicted targets of the regulated miRNAs. In addition, approximately one third of the significantly regulated mRNAs from the previous microarray study were identified as potential targets of the significantly changed miRNAs. miRNAs are key regulators of gene expression but the functions of many human miRNAs are yet to be discovered. This study provides the first analysis of miRNA levels in human alcoholism and raise the possibility that the reduced of expression of brain genes seen in human alcoholism may be due, at least in part, to increased levels of miRNAs. Supported by NIH AA12404.