Sravani Musunuri

Quantification of the Brain Proteome in Alzheimer’s Disease Using Multiplexed Mass Spectrometry

We have compared the brain proteome in the temporal neocortex between Alzheimers disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

Neuroproteomics tools in clinical practice.

Neurodegenerative disorders such as Alzheimers disease (AD), Parkinsons disease (PD), and amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Increased Levels of Extracellular Microvesicle Markers and Decreased Levels of Endocytic/Exocytic Proteins in the Alzheimer's Disease Brain

BACKGROUND Alzheimers disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-β and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins. OBJECTIVE This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology. METHODS Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays. RESULTS We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation. CONCLUSIONS Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.

Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins

Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature‐induced phase fractionation with the nonionic surfactant Triton X‐114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two‐dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In‐solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano‐LC and analyzed using two different high performance mass spectrometers, linear ion trap‐Fourier transform and a linear ion trap‐Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap‐Fourier transform and linear ion trap‐Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

Scavenger Receptor A Mediates the Clearance and Immunological Screening of MDA-Modified Antigen by M2-Type Macrophages

In this study, we investigated the uptake of malondialdehyde (MDA)-modified myelin oligodendrocyte glycoprotein (MOG) in the context of lipid peroxidation and its implications in CNS autoimmunity. The use of custom-produced fluorescently labeled versions of MOG or MDA-modified MOG enabled us to study and quantify the uptake by different macrophage populations and to identify the responsible receptor, namely SRA. The SRA-mediated uptake of MDA-modified MOG is roughly tenfold more efficient compared to that of the native form. Notably, this uptake is most strongly associated with anti-inflammatory M2-type macrophages. MDA-modified MOG was demonstrated to be resistant to degradation by lysine-dependent proteases in vitro, but the overall digestion fragments appeared to be similar in cell lysates, although their relative abundance appeared to be altered as a result of faster uptake. Accordingly, MDA-modified MOG is processed for presentation by APCs, allowing maximized recall proliferation of MOG35-55-specific 2D2 T cells in vitro due to higher uptake. However, MDA modification of MOG did not enhance immune priming or disease course in the in vivo MOG-EAE model, but did induce antibody responses to both MOG and MDA adducts. Taken together our results indicate that MDA adducts primarily constitute clearance signals for phagocytes and promote rapid removal of antigen, which is subjected to immunological screening by previously licensed T cells.

Nitration of MOG diminishes its encephalitogenicity depending on MHC haplotype

Post-translational modifications of autoantigens are hypothesized to affect their immunogenicity. We here report that nitration of tyrosine 40 in Myelin Oligodendrocyte Glycoprotein (MOG) abrogates its encephalitogenicity both at protein and peptide levels in the experimental autoimmune encephalomyelitis (EAE) model in H2b C57BL/6 mice. Furthermore, nitrated MOG displays inferior antigen-specific proliferation of 2D2 splenocytes in vitro. Conversely, H2q DBA1 mice remain fully susceptible to EAE induction using nitrated MOG as the dominant epitope of H2q mice is unaltered. Molecular modeling analysis of the MOG35-55/H2-IAb complex and bioinformatics peptide binding predictions indicate that the lack of T cell reactivity towards nitrated MOG can be attributed to the inability of murine H2-IAb to efficiently present the altered peptide ligand of MOG35-55 because the nitrated tyrosine 40 cannot be accommodated in the p1 anchor pocket. In conclusion we demonstrate nitration as a relevant determinant affecting T cell recognition of carrier antigen depending on MHC haplotype. Our data have implications for understanding the role of post-translationally modified antigen in autoimmunity.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Corrigendum to “Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways” [Free Rad. Biol. Med. (2017) 421–431]

Corrigendum to “Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways” [Free Rad. Biol. Med. (2017) 421–431]