Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Abstract

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.

FIGURE 1.

Identification of T46I mutation in a FALS family. A, the gene screening identified a point mutation T46I in VAPB. B, Thr⁴⁶, which is indicated by an arrowhead, is within the 16-amino acid fragment that is required for VAPB function in yeast, which is highlighted, and is highly conserved in VAP proteins from yeast to humans. C, sequences of human VAPB (hVAPB) protein and its Drosophila ortholog DVAP were aligned by using the ClustalW version 1.82 alignment program available from EMBL-EBI. Colon, an identity match; period, a conserved substitution according to the GONNET 250 matrix. The predicted functional domains in both proteins are preserved: a transmembrane domain (green) at the C terminus, a coil-coiled domain (red) in the middle, and a domain (gray) at the N terminus showing a significant homology to the nematode major sperm protein (MSP).

FIGURE 2.

Protein properties are modified by T46I. A, COS-7 cells were transfected with the indicated plasmids and fixed 48 h after transfection. The distribution of VAPB protein is indicated by GFP (green), and nuclei are shown with DAPI staining (blue). Scale bar, 20 μm. B, NSC-34 cells were harvested 48 h after transfection with empty GFP vector or GFP VAPBs. The separation of Triton X-100-soluble and insoluble fraction was carried out as described by Kanekura et al. (13). The fractions were blotted with anti-GFP antibody. The densitometry measurements of bands for soluble and insoluble VAPB are shown in C. Values are means ± S.E. (error bars) for three separate experiments.

FIGURE 3.

VAPB participates in the UPR. NSC-34 cells co-transfected with XBP1-Venus and RFP VAPB without (A) or with tunicamycin treatment (2 μg/ml, treated for 6 h before harvested) (B). RFP- and Venus-positive cells were quantified by FACS. The proportions as a percentage of Venus-positive cells in RFP-expressing cells are shown. C, cells stably expressing wild type or T46I-VAPB were transfected with Venus and RFP constructs. 24 h after transfection, cells were harvested and analyzed as in A and B. The p values from one-way ANOVA are 0.001 (A), 0.0035 (B), and 0.0417 (C), respectively. Bonferroni's multiple comparison test was applied as a post-test, and the significance values are shown in the graphs. D and E, the level of phosphorylated eIF2α was examined in transiently transfected cells (D) and in VAPB-stable expression cell lines (E) with or without thapsigargin treatment (500 nm for 30 min before harvest). After correcting with the loading control, actin, the level of phosphorylated eIF2α was quantified. All experiments were repeated at least three times. Values are means ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Mutant VAPB induces ubiquitin aggregate formation in both mutant protein-expressing and neighboring cells. A, 3 days after transfection with either vector or GFP-VAPBs constructs, NSC-34 cells were fixed and stained for endogenous ubiquitin. Examples of ubiquitin aggregates are indicated with arrows in each section. Ubiquitin aggregates found in GFP-negative cells are also pointed out with open arrowhead. The number of GFP-positive cells (B) and GFP-negative cells (C) containing ubiquitin aggregates was counted for three independent transfections. The p values from one-way ANOVA test are 0.0047 and 0.022, respectively. Bonferroni's multiple comparison tests were applied as a post-test, and the significance is shown in the graphs. Values are means ± S.E. (error bars). *, p < 0.05; **, p < 0.01.

FIGURE 5.

The expression of T46I-VAPB leads to a greater level of cell death. NSC-34 cells that stably express either wild type or T46I-VAPB were harvested for annexin V and 7-AAD staining after 3 days of culture. A, a representative experiment demonstrating the use of FACS to quantify the annexin V-positive cells. The gating of annexin V-positive cells and the percentage the gated cells are displayed. B, the annexin V- and 7-AAD-positive cells from four experiments were quantified. The p values from unpaired t test are 0.0038 and 0.0315 for annexin V and 7-AAD, respectively. C, a total of 3,000 cells were plated in each condition, whereas equal amounts of wild type- and T46I-VAPB-expressing cells were seeded in the co-culture. Cells were harvested and stained for annexin V 3 days after plating. Values are means ± S.E. (error bars) for three separate experiments. The p value from the one-way ANOVA test is 0.0004, and Bonferroni's multiple comparison test was applied as a post-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Targeted expression of T48I-DVAP in neurons induces the formation of aggregates immunoreactive for DVAP. A–C, neuronal cell bodies of WT larval brains immunostained with an anti-DVAP antibody (red) and anti-HRP antibody (green), a neuronal cell surface marker. DVAP is located underneath the cell membrane and in the cytoplasm. D–F, immunostaining with an anti-KDEL antibody, an ER-specific marker, shows that the cytoplasmic DVAP immunoreactivity associates with the ER. G–J, brains and nerve fibers of third instar larvae stained with antibodies for DVAP (red) and with antibodies for the neuronal cell surface marker anti-HRP (green). In controls (G), DVAP staining appears dispersed throughout the cytoplasm of neuronal cell bodies, whereas in T48I-DVAP transgenic brains (H), DVAP immunoreactivity is associated with intracellular aggregates of variable size. Nerve fibers of control larvae (I) and transgenic larvae (J) were stained with anti-HRP (green) and anti-DVAP antibodies (red). In control nerves, a faint and uniform staining for DVAP is observed, whereas in transgenic nerves, large aggregates strongly immunoreactive for DVAP accumulate along the nerves. Scale bars, 20 μm (J) and 10 μm (C, F, and H).

FIGURE 7.

Transgenic expression of T48I-DVAP in the adult fly eyes causes a severe disruption of eye morphology. A and B, scanning electron micrographs of controls (A) and transgenic adult fly eyes expressing the UAST48I-DVAP transgene under the control of the eye-specific driver, eyeless-GAL4 (B). C and D, higher magnifications of A and B, respectively. Whereas the adult Drosophila eye is composed of an ordered array of ommatidia and interspersed bristles, transgenic eyes appear smaller, with missing or extra bristles and fused ommatidia. Fused ommatidia are indicated by a white arrowhead, whereas a black arrowhead points to ommatidia with extra bristles. The white arrow points to missing bristles. Frontal sections of fly heads of control animals (E) and from flies expressing T48I-DVAP in the eye (F) are shown. Transgenic eyes show a severely disrupted internal morphology, cell degeneration, and numerous vacuoles (see arrowheads, for instance). G, summary of the relative frequency of eye sizes for WT and transgenes. H, quantification of the eye surface area showing that in transgenic flies expressing T48I-DVAP in the eye, the surface area is, on average, 50% smaller than controls. Scale bars, 200 μm (A and B) and 100 μm (C and D). Error bars, S.E.

FIGURE 8.

Transgenic expression of T48I-DVAP induces formation of aggregates composed of wild type and mutant proteins, ER fragmentation, and Hsp70 up-regulation. A–G, COS7 cells transfected with DVAP-FLAG (A) or FLAG-DVAP and MycT48I-DVAP (B–G) were stained with antibodies specific for the two tags. H–M, brains from control (H–J) and T48I-DVAP transgenic (K–M) larvae were stained for the ER marker, KDEL (green), and DVAP (red). N–S, brains from control (N–P) and T48I-DVAP transgenic (Q–S) larvae were stained for DVAP (red) and Hsp70 (green). Scale bars, 10 μm.

FIGURE 9.

Muscle-specific expression of T48I-DVAP causes cellular stress characterized by aggregate deposition, fragmentation of the sarcotubular system, and Hsp70 up-regulation. A–F, body wall muscle fibers from control larvae were stained for SERCA (green), as ER-sarcoplasmic reticulum marker and DVAP (red). A higher magnification of a smaller area is shown in D–F, demonstrating a high degree of overlap. G–L, control (G–I) and transgenic (J–L) muscles were stained for DVAP (red) and the ER marker, Boca (green). M–R, control (M–O) and transgenic (P–R) muscles were stained for DVAP (red) and the Hsp70 (green). Scale bars, 10 μm.

FIGURE 10.

The rare allele frequency comparison between normal and low VAPB expression groups. A, the positions of the six SNPs within the VAPB gene are shown. The whole VAPB gene, containing six exons (represented with filled boxes), spans to 57.5 kb. Five SNPs closest to each exon and one in the 3′-UTR (represented with an open box) were genotyped. B, the rare allele frequencies of the six SNPs are shown where the normal VAPB expression group is represented as filled bars, and the low VAPB expression group is represented with open bars. The group with normal VAPB expression level is composed of 16 SLAS, 1 FALS, and 19 controls, whereas the group with low VAPB expression level is composed of 24 SALS, 1 FALS, and 2 controls. Fisher exact test was applied for each SNP, and a significant difference of SNP rs6100067 was found (p = 0.029).

FIGURE 11.

Cellular disturbances triggered by mutant VAPB. In this study, we demonstrated (highlighted in boldface type) that the expression of ALS-linked mutant VAPB triggers VAPB protein aggregation as well as wild type protein redistribution. As a consequence, structure and function of ER are disrupted, which, together with the inability to activate the unfolded protein-responsive pathway, IRE1/XBP1, leads to potentiation of ER stress, the accumulation of ubiquitinated aggregates, and, eventually, cell death.