Proteomic Analysis of Lymphoblastoid Cells from Nasu-Hakola Patients: A Step Forward in Our Understanding of This Neurodegenerative Disorder

<div><p>Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer’s disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.</p></div

Two-dimensional electrophoretic map of proteins in Lymphoblastoid B-cells from individuals belonging to the family described in <b>Figure 1</b>.

<p>The virtual image reported here (Higher Master Gel, HMG), results from the correlation of master gels from each group of subjects and contains all the common and uncommon spots among groups of comparison. Spots differentially expressed among groups of subjects have been labeled by arrows with letters and numbers. Proteins contained in altered spots indicated by letters (<b>a</b> to <b>h</b>) were identified by gel-matching; those in spots arbitrarily indicated by numbers (1 to 13), by LC-MS/MS. For additional experimental details, see the text.</p

Up- and down-regulated proteins found in altered spots of our study, together with the values of their oscillation in expression level.

<p>Reference numbers refer to reports previously published describing alterations of these proteins in different neurodegenerative diseases.</p>a<p>p>0.05; ^bp<0.05.</p><p>Up- and down-regulated proteins found in altered spots of our study, together with the values of their oscillation in expression level.</p

Osteoclast pathway map found in KEGG pathway database.

<p>The red dotted circle indicates the cascade activated by SYK.</p

List of proteins identified under the immunoreactive spot.

<p>List of proteins identified under the immunoreactive spot.</p

Set of panels showing the density variances between SG and OT pools for spots 1 to 10.

<p>In each panel the region of the stained gel containing the spot of interest was magnified (inset) and the up-/downregulation graphically represented. Pvalue indicating statistical significant density variance (T-test) is reported in each panel.</p

(A) 2-DE map of OT obtained by performing IEF on a 4–7 linear pH range and SDS-PAGE on a constant 12,5% T in the second dimension, to separate proteins clustered in the single spot shown in Fig 4. (B) Immunoblotting of the gel slab indicated in Panel A.

<p>Arrow points to spot originated from separation and identified as FliD.</p

Immunoblotting of proteins from SG and OT profiles generated as indicated in Fig 1.

<p>PVDF membranes were incubated with the rabbit polyclonal antibodies anti-FliD of <i>M</i>. <i>mitochondrii</i>, followed by anti-rabbit antibody. The protein spot(s) indicated by an arrow in Panel A (OT pool) was tentatively assigned to FliD. Panel B shows the SG profile in which the hypothetical FliD spot is undetectable.</p

High Master gel, showing qualitative differences between the SG and OT 2-DE master gel patterns (NL, pH 3–10 gradient range).

<p>Labelled in green: spots (n = 170 ± 25) common to both SG and OT. Labelled in red: spots (n = 81) exclusive of SG. Labelled in blue: spots (n = 57) detected solely in OT.</p