VAPB ER-Aggregates, A Possible New Biomarker in ALS Pathology

Abstract

A point mutation (P56S) in the gene-encoding vesicle-associated membrane-protein-associated protein B (VAPB) leads to an autosomal-dominant form of amyotrophic lateral sclerosis (ALS), classified as ALS-8. The mutant VAPB is characterized by ER-associated aggregates that lead to a complete reorganization of ER structures. Growing evidences suggest VAPB involvement in ALS pathomechanisms. In fact, numerous studies demonstrated VAPB alteration also in sporadic ALS (sALS) and showed the presence of its aggregates when others ALS-related gene are mutant. Recently, the identification of new biomarkers in peripheral blood mononuclear cells (PBMCs) has been proposed as a good noninvasive option for studying ALS. Here, we evaluated VAPB as a possible ALS pathologic marker analyzing PBMCs of sALS patients. Immunofluorescence analysis (IFA) showed a peculiar pattern of VAPB aggregates in sALS, not evident in healthy control (HC) subjects and in Parkinson's disease (PD) PBMCs. This specific pattern led us to suppose that VAPB could be misfolded in sALS. The data indirectly confirmed by flow cytometry assay (FCA) showed a reduction of VAPB fluorescent signals in sALS. However, our observations were not associated with the presence of a genetic mutation or altered gene expression of VAPB. Our study brings further evidences of the VAPB role in ALS as a diagnostic biomarker.

Keywords: ALS; PBMC; VAPB ER-aggregates; biomarker; endoplasmic reticulum; flow cytometry; immunofluorescence.

Figure 1

Schematic representation of the vesicle-associated membrane-protein-associated protein B (VAPB). The arrow indicates the recognition site for an anti-VAPB monoclonal antibody (2-130 aa) used for flow cytometry assays (FCAs). This site includes the N-terminal domain (8-111 aa) of the protein, called MSP that can be affected by P56S mutation.

Figure 2

Representative immunofluorescence images performed with an antihuman VAPB polyclonal antibody in Hela cell lines. (a) VAPB (green) and nuclei (blue) in HeLa cells transfected with VAPB-Wt (left) and VAPB-P56S (right). Magnification is the same in both pictures (scale bar: 15 µm). The image shows VAPB aggregates and ER disorganization in VAPB-P56S-transfected HeLa cells caused by VAPB misfolding. (b) FCAs performed with an antihuman VAPB monoclonal antibody. The data, expressed as medium intensities of fluorescence (MFIs), show a statistically significant reduction of fluorescence signals in VAPB-P56S-transfected HeLa cells compared to in VAPB-Wt-transfected HeLa cells. (c) Representative western blot analysis showing the expression of GRP78, p62, and LC3 proteins and ubiquitin protein in VAPB-Wt- and VAPB-P56S-transfected cells. β-tubulin was used as a loading control. For each analysis, around 15 µg of protein were loaded. All western blot experiments were performed in triplicate. (d) Quantification of band intensities normalized with a β-tubulin value depicted in (c). Densitometry analysis was performed using the software ImageJ. Data are from one representative experiment out of three. *** p < 0.0005.

Figure 3

Representative confocal monochromatic nucleus (a,d,g), VAPB (b,e,h), and merge (c,f,i) images of immunofluorescence performed with an antihuman VAPB polyclonal antibody in PBMCs of HCs, PD patients, and sALS patients. VAPB is represented in green, and the nuclei are in blue. (i) The merge image shows the presence of VAPB ER-aggregates in sALS patients that is clearly different from those obtained in PD patients (f) and HCs (c). The arrows indicate VAPB ER-aggregates. Scale bars: 20 µm. Each sample were analyzed in 20 fields. For each subject, the number of lymphocytes showing VAPB aggregates was divided by the original total number of isolated cells (2 × 10⁴).

Figure 4

(a) Representative immunofluorescence image of primary skin fibroblasts performed with an antihuman VAPB polyclonal antibody. VAPB (green) and nuclei (blue) in fibroblasts were isolated from HCs (left) and sALS patients (right). The sALS patient fibroblasts staining showed globular accumulations of VAPB (indicated by arrows) compared to the control. (b) Image showing single staining of VAPB (green) (left), GRP78 (red) (middle), and merge (right). The merge evidenced the colocalization of GRP-78 with the VAPB accumulations (indicated by arrows) representing the ER distribution. Scale bars: 15 µm.

Figure 5

FCAs performed with a monoclonal antibody against VAPB. The graph shows a statistically significant decreased level of VAPB fluorescence detection in sALS patients compared to in PD patients and HCs. On the contrary, VAPB fluorescence signals in HCs and PD patients did not present significant differences. The data are expressed as MFIs in all patients and controls analyzed. * p < 0.05; ** p < 0.005.

Figure 6

VAPB mRNA expression in PBMCs from HCs (n = 24) and ALS (n = 24) patients. mRNA levels were quantified by real-time PCR. mRNA values are normalized by that of GAPDH. Values represent mean ± SD. Each sample was examined in triplicate. VAPB mRNA levels of sALS patients were not significantly different from those detected in HCs.

Figure 7

(a) Representative western blot data from one HC and two PD- and two sALS-affected patients. The image shows the overexpression of ubiquitin and HSP90 in sALS and PD patients compared to in the HCs. For each analysis, around 15 µg of protein were loaded. β-tubulin was used as a loading control. (b) Quantification of band intensities normalized with a β-tubulin value depicted in (a). Densitometry analysis was performed using the software Image. All western blot experiments were performed in triplicate. *** p < 0.0005.