Novel Exosome Biomarker Candidates for Alzheimer's Disease Unravelled Through Mass Spectrometry Analysis.

Exosomes are small extracellular vesicles (EVs) present in human biofluids that can transport specific disease-associated molecules. Consequently blood-derived exosomes have emerged as important peripheral biomarker sources for a wide range of diseases, among them Alzheimer's disease (AD). Although there is no effective cure for AD, an accurate diagnosis, relying on easily accessible peripheral biofluids, is still necessary to discriminate this disease from other dementias, test potential therapies and even monitor rate of disease progression. The ultimate goal is to produce a cost-effective and widely available alternative, which can also be employed as a first clinical screen. In this study, EVs with exosome-like characteristics were isolated from serum of Controls and AD cases through precipitation- and column-based methods, followed by mass spectrometry analysis. The resulting proteomes were characterized by Gene Ontology (GO) and multivariate analyses. Although GO terms were similar for exosomes' proteomes of Controls and ADs, using both methodologies, a clear segregation of disease cases was obtained when using the precipitation-based method. Nine significantly different abundant proteins were identified between Controls and AD cases, representing putative biomarker candidate targets. Among them are AACT and C4BPα, two Aß-binding proteins, whose exosome levels were further validated in individuals from independent cohorts using antibody-based approaches. The findings discussed represent an important contribution to the identification of novel exosomal biomarker candidates useful as potential blood-based tools for AD diagnosis.

Toward Neuroproteomics in Biological Psychiatry: A Systems Approach Unravels Okadaic Acid-Induced Alterations in the Neuronal Phosphoproteome.

Neuroproteomics is an evolving field of postgenomic medicine, highlighting the convergence of psychiatry/neurology and proteomics, yet compared with neurogenetics, it has received little attention. This study in rat primary neuronal cultures provides an example of a neuroproteomic approach relevant to the study of psychiatric disease pathophysiology, focusing on Alzheimer's disease. In this context, okadaic acid (OA) is routinely used in experimental designs to investigate phosphorylation-mediated events. It is a potent protein phosphatase (PP) inhibitor, particularly of PP1 and PP2A. Typically, a single protein and its phosphorylation level are monitored upon OA exposure. Although useful, this can be misleading as protein phosphorylation-mediated events involve complex signaling cascades and an array of kinases, phosphatases, and substrates. Bearing in mind the involvement of multiple pathways and cascade cross talk, this study employed a systems approach to analyze OA-induced molecular responses through PP inhibition. We showed that upon OA exposure, the recovery rate of 245 phosphoproteins significantly increased, while that of 75 significantly decreased. The prominent biological processes affected included anatomical structural development, transport, cell differentiation, and signal transduction. The associated phosphointeraction networks identified nodes representing OA-responsive phosphoproteins. Many of these are key players of signaling cascades relevant to a range of pathologies. In summary, the data presented results from a neuroproteomic preclinical study offering an array of phosphoproteins as potential targets for future diagnostic and therapeutic strategies in biological psychiatry. We note, however, the nonspecificity of targeting PPs themselves and emphasize the need for future neuroproteomic approaches toward systems psychiatry.

Altered protein phosphorylation as a resource for potential AD biomarkers.

The amyloidogenic peptide, Aß, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aß inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aß. In fact several Alzheimer's disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aß, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aß exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aß treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aß addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued.