Abstract
Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1-beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1(G93A) and TDP43(A315T). Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity.
Highlights
From the ‡Sagol School of Neuroscience and Department of Physiology and Pharmacology, Sackler School of Medicine and §Blavatnik School of Computer Science, Tel Aviv University, Israel; ¶Department of Biochemistry and Molecular Biology, the Uniformed Services University of Health Sciences, Bethesda, MD; Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, the Netherlands
In order to do so, we examined the distribution of Staufen1 and phosphatase 1– beta (PP1B) at the neuromuscular junction (NMJ), a specialized synapse structure comprised of the motoneuron axon, the muscle, and terminal Schwann cells
In the mSOD1 mouse model for Amyotrophic lateral sclerosis (ALS), mass spectrometry identified the phosphatase PP1B, whose connection to dynein was predicted based on the interaction of both proteins with the RNA-binding protein Staufen1
Summary
Mice—SOD1G93A mice [28] were originally obtained from Jackson Laboratories, and the colony was maintained by breeding with C57BL mice. Following overnight incubation with primary antibody, complexes were incubated with protein A agarose beads for 2 h at 4 °C and precipitated and washed with PBS with 0.1% Triton X-100 (Sigma). Western Blotting—Brain tissues were excised and homogenized in lysis buffer containing PBS, 1% Triton X-100 (Sigma), and protease inhibitors (Roche), followed by centrifugation and collection of the supernatant. RNA Immunoprecipitation—Total protein was harvested from brains of p60 female control and mSOD1 mice in extraction buffer (50 mM Tris-HCl, pH 7.5, 15 mM EGTA, 100 mM NaCl and 0.1% Triton X-100 supplemented with 1x protease inhibitors mixture (Roche Applied Sciences, Pensberg, Germany) and 10 unit/ml of RNAsin (Promega, Madison, WI)). Cover slides were washed in PBS and permeabilized with 0.5% Triton in blocking solution containing 5% donkey serum (Jackson Laboratories) and 1 mg/ml BSA (Sigma) for 5–30 min.
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