Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

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. Molecular & Cellular

Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

“…2b). In support, some glycolytic enzymes have also been identified in other proteomic studies on axonal transport-associated fractions92829. Usually considered as mere contaminants, some of the glycolytic enzymes might associate to specific structures, to efficiently provide the ATP needed for certain functions630.…”

The m6A‐epitranscriptomic signature in neurobiology: from neurodevelopment to brain plasticity

“…Indeed, biochemical subcellular fractionation or immunocytochemical assays have revealed the presence of m6A‐writers (METTL3, METTL14), erasers (FTO, ALKBH5), and readers (YTHDF1‐3) in the extra‐somatic regions of neurons (Gershoni‐Emek et al . ; Merkurjev et al . ; Yu et al .…”

“…The widespread existence of m6A-modified transcripts in regions distal from the neuronal cell body indicates an intriguing mode of m6A regulation outside the nucleus. Indeed, biochemical subcellular fractionation or immunocytochemical assays have revealed the presence of m6A-writers (METTL3, METTL14), erasers (FTO, ALKBH5), and readers (YTHDF1-3) in the extra-somatic regions of neurons (Gershoni-Emek et al 2016;Merkurjev et al 2017;Yu et al 2018). This not only allows the recognition of m6Amodified transcripts by cytoplasmic readers, but also the dynamic regulation of m6A outside the nucleus.…”

DYNLRB1 is Essential for Dynein Mediated Transport and Neuronal Survival

“…Our findings show that Dynlrb1 is critical not only for embryonic development, but also for maintenance of sensory neurons in adulthood. Deficits in dynein functions have been implicated in the pathogenesis of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) [40][41][42][43][44][45], and an increasing number of human neurodegenerative pathologies have been linked to mutations of dynein complex genes [46]. In addition, mutations and/or disruption of the function of the dynactin complex has been shown to be connected to motor neuron degeneration and ALS [47][48][49][50][51].…”