An integrated proteomic and bioinformatic approach for identification of Alzheimer's disease early biomarkers

Motivation: An increase in human life span is expected to double in the next 30 years. As the elderly population expands, the prevalence of neurodegenerative diseases such as Alzheimer's disease (AD) or other forms of dementia is likely increasing, inducing consequently profound economic and social implications. Thirty-five million people are now considered to be affected by AD and this number is expected to double in the next few decades. Consequently, AD is now considered a very serious public health problem mainly for the direct and indirect costs of their care. One of the histopathological hallmarks of AD is the amyloid plaques formation in the brain. The plaques are mainly constituted by aggregates of A? peptide, a peptide generated by a proteolytic cleavage of the amyloid precursor protein (APP). However, the early events in the onset of the pathology are yet to be fully explained and the mechanisms through which A? triggers the cascade of events leading to AD are unknown. Alterations in proteomic pathway have been described in many neurodegenerative diseases, including in AD. The aim of this study was identified potential biomarkers involved in early AD stage by using proteomic approach and bioinformatic tools. For this purpose, we experimentally mimed AD early stage conditions by using neuronal cells treated for short time with A? peptide. Methods: LAN5 neuroblastoma cells were incubated without (control) or with A? peptide for a short time (1 hour) at 37°C. Proteins obtained from these cells were analyzed with a shotgun proteomics approach by LTQ-Orbitrap-MS followed by label-free quantitation. Significantly up- and down-regulated proteins were identified by volcano plot STRING was used to visualize protein-protein interaction between the proteins identified as modulated. DAVID and REViGO were used for GO annotation. LAN5 protein expression after A? treatment was analyzed by Western blot incubated with specific antibodies. Results: An in vitro AD early stage was simulate by treating LAN5 cells with A? for short time. Protein extracted from untreated and treated cells were subjected to shotgun proteomic analysis followed by bioinformatics approach. A volcano plot was built from the mass spectrometry data for visualizing significantly modulated proteins (p < 0. 05 and log2- fold change <-0.5 or log2 > 0.5). A total of 159 proteins were found to be up-regulated and 88 to be down-regulated relative to the control. By STRING and GO annotation two protein-protein interaction networks were identified. The first network contained proteins related to rRNA metabolism and the second one consisted mainly of proteins related to RNA metabolism, and many with the splicing process. The pathway enrichment identified using KEGG mainly showed up-regulation of the lysosomal pathway and down regulation of the spliceosomal pathway. To confirm that A? inhibits expression of proteins involved in the spliceosomal pathway the modulation of expression of SmB /B'/N and PRP6, two ubiquitous proteins of the spliceosome, was analyzed. By Western blot incubated with specific antibodies we confirmed down regulation of these proteins in a time-dependent manner. In conclusion, this study provides evidence that a short exposure of LAN5 with A? can be considered a model to mimic early AD stages. Several up- and down-regulated proteins involved in different dysfunction occurring in AD have been found as a consequence of A? stimulation. In particular, down-regulation of spliceosome components suggests that the modification of the splicing machinery is a key event in early AD and the proteins involved can be utilized as potential biomarkers.