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. 2012 May 1;21(9):1979-88.
doi: 10.1093/hmg/dds011. Epub 2012 Jan 17.

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria

Gábor M Mórotz  1 , Kurt J De Vos, Alessio Vagnoni, Steven Ackerley, Christopher E Shaw, Christopher C J Miller
Affiliations

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria

Gábor M Mórotz et al. Hum Mol Genet. .

Abstract

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca(2+)]c); elevated [Ca(2+)]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca(2+)]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca(2+)]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca(2+) insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca(2+) homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

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Figures

Figure 1.
Figure 1.
VAPBP56S disrupts anterograde axonal transport of mitochondria. Mitochondrial transport was recorded in neurons co-transfected with DsRed-Mito and either EGFP (CTRL), EGFP-VAPB (VAPB) or EGFP-VAPBP56S (VAPBP56S) as indicated. (A) Representative kymographs show axonal transport of mitochondria in EGFP, VAPB or VAPBP56S transfected neurons. (B) The percentages of motile, anterograde and retrograde moving mitochondria are shown. Expression of VAPB had no effect on mitochondrial transport. In contrast, VAPBP56S reduced mitochondrial transport and this was due to a selective inhibition of anterograde but not retrograde transport. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. n = 11–14 cells; error bars are SEM. **P < 0.01; ***P < 0.001.
Figure 2.
Figure 2.
VAPBP56S reduces the amount of endogenous tubulin but not endogenous kinesin-1 associated with Miro1. (A) HEK293 cells were co-transfected with either empty vector (EV), empty vector + Myc-Miro1 + HA-TRAK1, VAPB + Myc-Miro1 + HA-TRAK1 or VAPBP56S + Myc-Miro1 + HA-TRAK1 as indicated. Miro1 was immunoprecipitated using the myc-tag and the amounts of co-immunoprecipitating kinesin-1 and α-tubulin detected by immunoblotting. Samples of the input lysates (Input) and immunoprecipitates (IP:Myc-Miro1) are shown. (B) Bar graphs show the relative levels of kinesin-1 and α-tubulin in the immunoprecipitates. Kinesin-1 signals were normalized to immunoprecipitated myc-Miro1 signal; α-tubulin signals were normalized to both immunoprecipitated myc-Miro1 and immunoprecipitated kinesin-1 signals as indicated. Values were converted so that empty vector + Myc-Miro1 + HA-TRAK1 transfection was assigned a reference value of 1.0. The results shown are from four independent transfections. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. n = 4; error bars are SEM. *P < 0.05; **P < 0.01.
Figure 3.
Figure 3.
Expression of VAPBP56S increases resting [Ca2+]c levels in neurons. Neurons were transfected with either control EGFP vector, EGFP-VAPB (VAPB) or EGFP-VAPBP56S (P56S) and [Ca2+]c determined by Fura2 ratio imaging. Resting [Ca2+]c was calculated for each individual neuron as the average resting [Ca2+]c between 60 and 180 s of measurement (indicated with box in (A). Values for individual cells were then collated to generate the bar graph in (B). To ensure that resting [Ca2+]c was obtained from viable neurons, we induced a transient influx of Ca2+ by a 2 min application of 50 mm KCl to depolarize the neurons. Only cells that showed a transient increase in [Ca2+]c after depolarization were included in the analysis of resting [Ca2+]c. Statistical significance was determined by one-way ANOVA (Kruskal–Wallis) followed by Dunn's multiple comparison test. n = 10 (CTRL), 14 (VAPB) and 20 (VAPBP56S) cells; error bars are SEM. *P < 0.05.
Figure 4.
Figure 4.
Expression of Ca2+-insensitive Miro1E208K/E328K rescues the effect of VAPBP56S on the association of tubulin with Miro1. (A) HEK293 cells were co-transfected with either empty vector (EV), empty vector + myc-Miro1E208K/E328K (Myc-Miro1KK) + HA-TRAK1, VAPB + myc-Miro1E208K/E328K + HA-TRAK1 or VAPBP56S + myc-Miro1E208K/E328K + HA-TRAK1 as indicated. Miro1E208K/E328K was immunoprecipitated using the myc-tag and the amounts of co-immunoprecipitating kinesin-1 and α-tubulin detected by immunoblotting. Samples of the input lysates (Input) and immunoprecipitates (IP:Myc-Miro1KK) are shown. (B) Bar graphs show relative levels of kinesin-1 and α-tubulin in the immunoprecipitates. Kinesin-1 signals were normalized to immunoprecipitated myc-Miro1E208K/E328K signal; α-tubulin signals were normalized to both immunoprecipitated myc-Miro1E208K/E328K and immunoprecipitated kinesin-1 signals as indicated. Values were converted to so that empty vector + myc-Miro1E208K/E328K + HA-TRAK1 transfection was assigned a reference value of 1.0. The results shown are from three independent transfections. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. n = 3; error bars are SEM.
Figure 5.
Figure 5.
VAPBP56S-induced defective anterograde mitochondrial transport is rescued by expression of Ca2+-insensitive Miro1E208K/E328K. Neurons were co-transfected with DsRed-Mito and either EGFP-VAPB, EGFP-VAPBP56S, EGFP-VAPBP56S + Miro1 or EGFP-VAPBP56S + Miro1E208K/E328K. Transfections were balanced with empty vector so that all treatments received the same total amounts of DNA. (A) The proportion of anterograde moving mitochondria. Expression of EGFP-VAPBP56S (P56S) reduced anterograde mitochondrial transport and this was rescued by expression of Miro1E208K/E328K (MiroKK) but not wild-type Miro1. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. n = 15–17; error bars are SEM; *P < 0.05, **P < 0.01; ***P < 0.001, ns not significant. (B) Representative kymographs of mitochondrial movement in neurons co-transfected with EGFP-VAPB (VAPB), EGFP-VAPBP56S (VAPBP56S), EGFP-VAPBP56S + Miro1 (VAPBP56S + Miro1) or EGFP-VAPBP56S + Miro1E208K/E328K (VAPBP56S + Miro1KK) as indicated.
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References

    1. Hollenbeck P.J., Saxton W.M. The axonal transport of mitochondria. J. Cell Sci. 2005;118:5411–5419. - PMC - PubMed
    1. Glater E.E., Megeath L.J., Stowers R.S., Schwarz T.L. Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent. J. Cell Biol. 2006;173:545–557. - PMC - PubMed
    1. Pilling A.D., Horiuchi D., Lively C.M., Saxton W.M. Kinesin-1 and dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol. Biol. Cell. 2006;17:2057–2068. - PMC - PubMed
    1. Tanaka Y., Kanai Y., Okada Y., Nonaka S., Takeda S., Harada A., Hirokawa N. Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell. 1998;93:1147–1158. - PubMed
    1. Hurd D.D., Saxton W.M. Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila. Genetics. 1996;144:1075–1085. - PMC - PubMed

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