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PubMed2006

Novel differential neuroproteomics analysis of traumatic brain injury in rats.

Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and alphaII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.

Animals↗

PubMed2006

Potential inplications of endogenous aldehydes in beta-amyloid misfolding, oligomerization and fibrillogenesis.

Aldehydes are capable of inducing protein cross-linkage. An increase in aldehydes has been found in Alzheimer's disease. Formaldehyde and methylglyoxal are produced via deamination of, respectively, methylamine and aminoacetone catalyzed by semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6. The enzyme is located on the outer surface of the vasculature, where amyloidosis is often initiated. A high SSAO level has been identified as a risk factor for vascular disorders. Serum SSAO activity has been found to be increased in Alzheimer's patients. Malondialdehyde and 4-hydroxynonenal are derived from lipid peroxidation under oxidative stress, which is also associated with Alzheimer's disease. Aldehydes may potentially play roles in beta-amyloid aggregation related to the pathology of Alzheimer's disease. In the present study, thioflavin-T fluorometry, dynamic light scattering, circular dichroism spectroscopy and atomic force microscopy were employed to reveal the effect of endogenous aldehydes on beta-amyloid at different stages, i.e. beta-sheet formation, oligomerization and fibrillogenesis. Formaldehyde, methylglyoxal and malondialdehyde and, to a lesser extent, 4-hydroxynonenal are not only capable of enhancing the rate of formation of beta-amyloid beta-sheets, oligomers and protofibrils but also of increasing the size of the aggregates. The possible relevance to Alzheimer's disease of the effects of these aldehydes on beta-amyloid deposition is discussed.

Aldehydes↗

PubMed2002

Functional genomics approaches to understanding brain disorders.

The completed draft of the human genome sequence has facilitated a revolution in neuroscience research. This sequence information and the development of new technologies used to analyze gene expression on a genomic scale provides a new and powerful means to investigate brain disorders of unknown etiology and to isolate novel drug targets for these disorders. The term functional genomics broadly describes a set of technologies and strategies directed at the problem of determining the function of genes, and understanding how the genome works together to generate whole patterns of biological function. The most powerful of these functional genomics approaches, expression profiling or DNA microarrays, can be used to analyze the expression of thousands of genes simultaneously. The results to date from the application of DNA microarray methods to postmortem diseased human brain tissue, animal models and cell culture models of brain disorders provide an exciting glimpse into the future of this field.

Animals↗

PubMed2026

A spatially resolved genomic-molecular atlas of human white-matter microstructure.

Human white matter has been linked to inherited variation, circulating molecular state and brain disease, but these layers have rarely been mapped onto the same tract anatomy. Here we measured genetic effects along 6,090 atlas-aligned fiber pathways sampled at 609,000 locations in 72,185 UK Biobank participants, and integrated proteomic and metabolomic profiles within the same anatomical frame. Genetic effects were not whole-tract properties: each locus formed a spatial footprint along fiber trajectories, ranging from single locations to broad multi-tract patterns and reflecting regional polygenicity rather than tract heritability. This map identified 258, 186 and 298 previously unreported loci for fractional anisotropy, mean diffusivity and axial diffusivity; spatial patterns replicated in adults and 157 of 315 FA loci replicated in adolescence in ABCD. Mendelian randomization linked localized genetic effects to neurodegenerative and psychiatric traits, with Alzheimer's disease showing directional effects across 12 of 17 tracts. Multi-omic analyses identified 97 proteomic and 161 metabolomic associations, with the broadest signals from lipid metabolites including linoleic acid and phosphatidylcholines. The strongest lipid-metabolite and genetic signals converged in the corpus callosum, placing inherited variation, disease risk and systemic lipid metabolism on the same localized tract segments.

Journal Article↗

PubMed2005

Characterization of an RNA granule from developing brain.

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

Actins↗

PubMed2006

The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity.

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.

Animals↗

PubMed2005

Application of partial least squares discriminant analysis to two-dimensional difference gel studies in expression proteomics.

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling ith cyanine dyes. In multi-gel experiments, univariate statistical tests have been used to identify differential expression between sample types by looking for significant changes in spot volume. Multivariate statistical tests, which look for correlated changes between sample types, provide an alternate approach for identifying spots with differential expression. Partial least squares-discriminant analysis (PLS-DA), a multivariate statistical approach, was combined with an iterative threshold process to identify which protein spots had the greatest contribution to the model, and compared to univariate test for three datasets. This included one dataset where no biological difference was expected. The novel multivariate approach, detailed here, represents a method to complement the univariate approach in identification of differentially expressed protein spots. This new approach has the advantages of reduced risk of false-positives and the identification of spots that are significantly altered in terms of correlated expression rather than absolute expression values.

Analysis of Variance↗

PubMed2003

Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1)-associated dementia is a neuroinflammatory brain disorder that is fueled by viral infection and immune activation of brain mononuclear phagocytes (MP; macrophages and microglia). MP serve as a reservoir for persistent viral infection, a vehicle for viral dissemination throughout the brain, and a major source of neurotoxic products that when produced in abundance, affect neuronal function. Such neurotoxic substances secreted by MP lead to clinical neurological impairment (cognitive, behavior, and motor abnormalities), which occurs usually years after the initial viral infection. How HIV-1 evades the immune function characteristic for MP as a first line of defense, including phagocytosis and intracellular killing, is not well understood despite more than two decades of study. In this report, we review the complex role(s) played by MP in the neuropathogenesis of HIV-1 infection. The clinical manifestations, pathology and pathogenesis, and treatment options are discussed in relationship to innate and adaptive immunity. Particular emphasis is given to the diversity of MP functions and how it may affect the disease process and manifestations. New insights into disease mechanisms are provided by advances in enhanced magnetic resonance imaging and proteomics to identify cell movement and genetic profiles of disease. New therapeutic strategies are discussed based on current knowledge of HIV-1-associated dementia pathogenesis.

AIDS Dementia Complex↗

PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗

PubMed2003

Peptidomics in Drosophila melanogaster.

In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analysed, the peptidomic approach aims at the simultaneous visualisation and identification of the whole peptidome of a cell or tissue, ie all expressed peptides with their post-translational modifications. With nanoscale liquid chromatography (nanoLC), combined with mass spectrometry and subsequent database searching, the peptidome of the Drosophila larval brain has been identified at the amino acid sequence level. In a single experiment involving only 50 Drosophila larval brains, one can obtain a display of the expressed peptides. In this paper, current peptidomics technology will be explained, using Drosophila as an example. Compared with the 400,000 Drosophila whole bodies that were required as a starting material for traditional biochemical peptide purification rounds, the authors are convinced that peptidomics technology, which in the future will certainly be applied to the analysis of different physiological states, has the inherent potential to bring about a true revolution in the study of the molecular physiology of Drosophila.

Animals↗

PubMed2004

Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model.

The analysis of rapid changes of protein expression in living systems in response to insults requires rigorous methods of complexity reduction. To control dynamic pattern of hundreds or even thousands of protein isoforms, we applied a novel method of differential molecular analysis to a cellular model which is suited to study ischemia. Neural derivatives of murine embryonic stem cells were exposed to chemical ischemia. The model was used to obtain starting material for a quantitative differential proteomics analysis. Fractionation of phosphoproteins from these samples and subsequent identification by mass spectrometry of differential proteins provide proof of principle of how novel molecular analytical tools provide new insight into the network of neuroprotective molecular events during specific situations of neuronal stress and related pharmaceutical intervention. Our results indicate a particular role of an isoform of the acidic calcium-independent phospholipase A2 in this type of insult.

Alkylation↗

PubMed2003

A dual role for poly-ADP-ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14-3-3gamma.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a homeostatic enzyme that paradoxically contributes to disturbances in spatial memory acquisition after traumatic brain injury (TBI) in transgenic mice, thought to be related to depletion of its substrate nicotinamide adenine dinucleotide (NAD+). In this study, systemic administration of the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) after TBI preserved brain NAD+ levels and dose-dependently reduced poly-ADP-ribosylation 24 h after injury. While moderate-dose INH2BP improved spatial memory acquisition after TBI; strikingly, both injured- and sham-mice receiving high-dose INH2BP were unable to learn in the Morris-water maze. Poly-ADP-ribosylated peptides identified using a proteomics approach yielded several proteins potentially associated with memory, including structural proteins (tubulin alpha and beta, gamma-actin, and alpha-internexin neuronal intermediate filament protein) and 14-3-3gamma. Nuclear poly-ADP-ribosylation of 14-3-3gamma was completely inhibited by the dose of INH2BP that produced profound memory disturbances. Thus, partial inhibition of poly-ADP-ribosylation preserves NAD+ and improves functional outcome after TBI, whereas more complete inhibition impairs spatial memory acquisition independent of injury, and is associated with ribosylation of 14-3-3gamma.

14-3-3 Proteins↗

PubMedDate not supplied

Application of a systems biology approach to developmental neurotoxicology.

Systems biology can be applied to enhance the understanding of complex biological processes such as apoptosis in the developing brain. Systems biology, as applied to toxicology, provides a structure to arrange information in the form of a biological model. The approach allows for the subsequent and iterative perturbation of the initial model with the use of toxicants, and the comparison of the resulting data against the proposed biological model. It is postulated that the exposure of the developing rat to NMDA antagonists, e.g., ketamine or phencyclidine (PCP), causes a compensatory up-regulation of NMDA receptors, thereby making cells bearing these receptors more vulnerable to excitotoxic effects of endogenous glutamate. Although comprehensive gene expression/proteomic studies and mathematical modeling remain to be accomplished, a biological model has been established and perturbed in an iterative manner to allow confirmation of the biological pathway for NMDA antagonist-induced brain cell death in the developing rat.

Animals↗

PubMed2001

Proteomic comparison of human and great ape blood plasma reveals conserved glycosylation and differences in thyroid hormone metabolism.

Most blood plasma proteins are glycosylated. These glycoproteins typically carry sialic acid-bearing sugar chains, which can modify the observed molecular weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conservation of plasma protein expression and glycosylation during approximately 12 million years of evolution. A few lineage-specific differences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quantitative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cerebrospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed approximately 2-fold higher levels in chimpanzees compared to humans. Since transthyretin binds thyroid hormones, we next compared plasma thyroid hormone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Notably, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many organs and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulating delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explaining unique features of human evolution are discussed.

Animals↗

PubMed2004

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature.

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.

Aging↗

PubMed2003

Initial large-scale exploration of protein-protein interactions in human brain.

Study of protein interaction networks is crucial to post-genomic systems biology. Aided by high-throughput screening technologies, biologists are rapidly accumulating protein-protein interaction data. Using a random yeast two-hybrid (R2H) process, we have performed large-scale yeast two-hybrid searches with approximately fifty thousand random human brain cDNA bait fragments against a human brain cDNA prey fragment library. From these searches, we have identified 13,656 unique protein-protein interaction pairs involving 4,473 distinct known human loci. In this paper, we have performed our initial characterization of the protein interaction network in human brain tissue. We have classified and characterized all identified interactions based on Gene Ontology (GO) annotation of interacting loci. We have also described the "scale-free" topological structure of the network.

Brain↗

PubMed2004

High reproducibility of large-gel two-dimensional electrophoresis.

Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER trade mark. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.

Animals↗

PubMed2005

[Proteomic comparison between human cerebellums and frontal lobes].

OBJECTIVE: To compare the results of proteomics between the cerebellum and frontal lobe of the aged. METHOD: Proteins were isolated from human cerebellums and frontal lobes and separated by two-dimensional (2D) gel electrophoresis. The proteins were then stained with silver or colloidal coomassie blue to produce a high-resolution map of the proteome. Selected proteins from this map were in-gel digested with trypsin and the resulting tryptic peptides were analyzed by MALDI-TOF mass spectrometry and MALDI TOF/TOF tandem mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. RESULTS: Three up-regulated protein spots in 2D gels of cerebellums were found as compared with those of frontal lobes. These protein spots were identified as antioxidant protein 2, creatine kinase precursor, fructose-bisphosphate aldolase C. Two down-regulated proteins in cerebellums were identified as pyruvate kinase M1 isozyme and glial fibrillary acidic protein. 30 common proteins, the expressions of which were not altered between cerebellums and frontal lobes, were also identified. CONCLUSIONS: These data will be used for future studies of differential protein expression in neurological disorders. Some proteins are useful for discovering the molecular mechanisms of degenerative dementia and brain aging.

Aged↗