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. 2013 Jan 3:8:1.
doi: 10.1186/1750-1326-8-1.

Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice

Linghua Qiu  1 , Tao Qiao, Melissa Beers, Weijia Tan, Hongyan Wang, Bin Yang, Zuoshang Xu
Affiliations

Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice

Linghua Qiu et al. Mol Neurodegener. .

Abstract

Background: A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results: To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion: Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.

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Figures

Figure 1
Figure 1
Overexpression of wtVAPB or muVAPB in the spinal cord and brain of the transgenic mice. (A) Schematic diagram showing the transgene construct. This construct has a CAG promoter, which drives the expression of human VAPB gene that is tagged by 3XFLAG (marked as 3XFL) at its N-terminal. The 3X poly A sequences (3XpA) after the VAPB gene terminate the transcription. Therefore, the RFP gene is not expressed. The two triangles represent the loxP sequence that flank the VAPB transgene. (B) VAPB levels from two lines of muVAPB and one line of wtVAPB transgenic mice were determined by Western blot. For the detection of VAPB in the spinal cords, 100 μg protein was loaded in each lane. The proteins were resolved by a 12% SDS-PAGE and detected as described in the materials and methods. For the detection of VAPB in the brains, 30 μg protein was loaded in each lane. Due to its 3XFLAG tag, the molecular weight of the transgenic VAPB (as indicated by *) is slightly more than the endogenous mouse VAPB protein.
Figure 2
Figure 2
Overexpression of muVAPB, but not wtVAPB, causes aggregation. (A) Lumbar spinal cord sections from different transgenic lines (NTG, wtVAPB, muVAPB35, and muVAPB35/60) were stained with VAPB antibody. VAPB-positive inclusions were found throughout the whole spinal cord in the muVAPB mice but not in the wtVAPB mice. The inserts show high magnification of the boxed areas in the large image. Aggregates are indicated by arrows. Scale bars: 30 μm. (B) Motor neurons in muVAPB mice, but not in wtVAPB mice, show VAPB aggregates. The neurons were marked by staining for neurofilaments high molecular weight subunit (NF-H). The VAPB aggregates are indicated by arrows in the figure. Scale bars: 10 μm.
Figure 3
Figure 3
Age-associated changes in body weight and motor function in VAPB transgenic mice. (A) No significant difference in the body weight between female muVAPB and NTG mice up to 600 days old. For each time point, 6 to 21 mice from each genotype were analyzed. (B) No significant difference among muVAPB35/60 (n = 4), muVAPB35 (n = 11) and NTG (n = 11) mice in rotarod test at the age of 10 to 12 months. (C, D) Grip strength tests of the forelimbs (C) and combined forelimbs and hindlimbs (D) in female NTG and muVAPB35 transgenic mice at different ages. The number of mice tested from each genotype at each time point is in the range of 4 to 22. All values are displayed as averages with standard deviation.
Figure 4
Figure 4
No significant pathological change in 18-month old VAPB transgenic mice. Spinal cord sections (A) of the ventral horn area and L3 ventral root nerve sections (B) from different genotypes are shown. All plastic sections were cut at 1 μm thickness and stained with toluidine blue. Scale bars: 20 μm. (C) Numbers of total axons of the ventral roots (L3 and L4) from 18-month old mice were counted. No significant difference in the numbers of axons was found between muVAPB35 (n = 5) and NTG (n = 4) mice. All values are shown as averages with standard deviation. (D) No evidence of microgliosis and astrogliosis was detected in the spinal cord of muVAPB35/60 mice, as compared with NTG mice. The sections from the lumbar spinal cords were stained with Iba1 and GFAP antibodies (green fluorescence) to show microglia and astrocytes, respectively. The nuclei were shown by DAPI stain. Scale bars: 20 μm. (E) No pathological change in the muscles from transgenic mice. Gastrocnemius muscle sections from NTG, wtVAPB, and mutVAPB35/60 were stained with H&E. Scale bars: 50 μm.
Figure 5
Figure 5
No significant TDP-43 pathology and ubiquitin-positivity in the muVAPB transgenic mice. (A) No TDP-43 aggregation or change in its cellular distribution in the spinal cords of VAPB transgenic mice was detected. Frozen sections from lumbar spinal cords from NTG, wtVAPB and muVAPB35/60 mice at 18-month old were stained with VAPB and TDP-43 antibodies. Scale bars: 10 μm. (B) Ubiquitin staining was absent or weak in motor neurons with muVAPB aggregates. Frozen sections from cervical spinal cords from NTG and muVAPB35/60 at 18-month old were stained with ubiquitin and VAPB antibodies. The sections from mutant SOD1 mice at their end stage were stained in parallel as positive controls. Scale bars: 6 μm. The VAPB aggregates are indicated by arrows in the figure. The nuclei are shown by DAPI stain.
Figure 6
Figure 6
muVAPB did not affect ER integrity. Frozen sections from the lumbar spinal cords of NTG, wtVAPB and muVAPB35/60 mice at 18-month old were stained with VAPB and PDI antibodies. VAPB aggregates are indicated by arrows. Scale bars: 10 μm.
Figure 7
Figure 7
muVAPB did not cause structural change for ER and mitochondria or induce UPR response. (A) ER and mitochondrial morphology in the motor neurons from the lumbar spinal cords of muVAPB35/60 mice at 18-month old were not different from the NTG controls. (Abbreviations: Mit-mitochodria, Lf-lipofuscin, Nuc-nucleus, ER-endoplasmic reticulum.) Scale bars: 5 μm. (B) Relative ratios of Chop and BiP mRNA from the spinal cords of muVAPB35 mice at the age of 14 months as determined by RT-PCR and compared to the age-matched NTG mice. All values are represented by mean±SD. No significant difference was detected.
Figure 8
Figure 8
muVAPB did not cause coaggregation of VAPA. Sections from the lumbar spinal cords from NTG and muVAPB35/60 mice at 18-month old were stained for VAPA. NF-H was stained as a marker for neurons. The cell nuclei were shown by DAPI stain. Scale bars: 20 μm.
Figure 9
Figure 9
Overexpression of neither wtVAPB nor muVAPB significantly impacted on the disease progression and survival of SOD1G93A mice. (A) The effect of overexpression of muVAPB on the weight changes in SOD1G93A mice. The weight data of the male mice from SOD1G93A (n = 12), SOD1G93A and muVAPB35 double transgenic (n = 10), SOD1G93A and muVAPB35/60 triple transgenic mice (n = 10) were analyzed. (B) The survival of the male mice from SOD1G93A (n = 27), SOD1G93A and muVAPB35 double transgenic (n = 27), SOD1G93A and muVAPB35/60 triple transgenic (n = 27), and SOD1G93A and wtVAPB double transgenic mice (n = 6) were analyzed.
Figure 10
Figure 10
VAPBP56S and SOD1G93A aggregates did not interact with each other. (A). Mouse spinal cord lysates were centrifuged to separate the soluble and insoluble fractions. One hundred μg of both fractions were loaded on the gel and blotted for VAPB, FLAG, SOD1 and tubulin. The asterisks mark the 3XFLAG tagged VAPB, which is above the endogenous mouse VAPB. (B). muVAPB and SOD1G93A aggregate independent of each other in the same motor neuron in the spinal cord of the muVAPB35 and SOD1G93A double transgenic mice. The sections from the lumbar spinal cord were stained with VAPB and hSOD1 antibodies. The nuclei were shown by DAPI stain. Scale bar: 5 μm.
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