Proteomics to investigate the molecular bases of Nasu-Hakola Disease

1. Purpose. Nasu-Hakola disease (NHD), also known as Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy (PLOSL), is a recessively inherited rare systemic disorder characterized by simultaneous impairment of nervous and skeletal systems. The genetic bases of NHD have been related to a structural defect of two genes (DAP12 and TREM2) encoding for different subunits of the membrane receptor signaling complex, expressed by cells of both systems involved [1]. To date, molecular or proteomic studies about this disorder are scarce, and only case reports have been reported. Being the molecular analysis mandatory for obtaining insights into the mechanisms of the disease, aim of the present study was the development of an optimal proteomic approach to characterize the proteome of Lymphoblastoid cells from PLOSL patients. 2. Materials. Two NHD patients with a novel mutation in exon 2 of the TREM2 gene (NHD); four healthy carriers (HC) and two healthy subjects (HS) belonging to the same Italian family were submitted to blood withdrawal to isolate and immortalize the B-Lymphocytes used for this research [2]. 3. Methods. About 30x106 B-Lymphocytes were recovered from plasma samples and immortalized. Proteins from each sample were treated with a MS compatible detergent, digested by trypsin and analyzed using a gel-free proteomic approach (Multidimensional Protein Identification Technology, MudPIT), according to conditions previously reported [3]. Finally, data handling was performed using the 3.3.1 Bioworks and MAProMa softwares. 4. Results. MudPIT analysis allowed the identification of more than 3000 distinct proteins. The comparison of protein profiles from NHD; HC and HS groups by means of MAProMa showed that, while the three groups shared more than 50% of proteins, only 10% were peculiar of each category. Of note, the proteins differentially expressed among groups were in agreement with those previously characterized by applying a two-dimensional gel electrophoresis (2-DE) approach[2]. Identification of these proteins allowed to confirm that also NHD onset may involve the glucose pathway, as suggested for other neurodegenerative disorders. 5. Discussion & Conclusion. Application of MudPIT proteomics analysis to Lymphoblastoid cells of individuals investigated in this study allowed to characterize a number of proteins about 100-fold higher than those identified by 2-DE [2] and to highlight proteins associated to other neurodegenerative processes. Given the absence of reports dealing with the application of shotgun proteomics to NHD investigation, this work represents a proof of principle in this field. In fact, an extensive protein profile of Lymphoblastoid cells from NHD subjects has been obtained that will be useful to characterize metabolic pathways involved in functional alterations of this pathology.