Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1575-8) contains supplementary material, which is available to authorized users.

Mutations in the RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) cause amyotrophic lateral sclerosis (ALS). Although ALS-linked mutations in FUS often lead to a cytosolic mislocalization of the protein, the pathogenic mechanisms underlying these mutations remain poorly understood. To gain insight into these mechanisms, we examined the biochemical, cell biological and functional properties of mutant FUS in neurons. Expression of different FUS mutants (R521C, R521H, P525L) in neurons caused axonal defects. A protein interaction screen performed to explain these phenotypes identified numerous FUS interactors including the spinal muscular atrophy (SMA) causing protein survival motor neuron (SMN). Biochemical experiments showed that FUS and SMN interact directly and endogenously, and that this interaction can be regulated by FUS mutations. Immunostaining revealed co-localization of mutant FUS aggregates and SMN in primary neurons. This redistribution of SMN to cytosolic FUS accumulations led to a decrease in axonal SMN. Finally, cell biological experiments showed that overexpression of SMN rescued the axonal defects induced by mutant FUS, suggesting that FUS mutations cause axonal defects through SMN. This study shows that neuronal aggregates formed by mutant FUS protein may aberrantly sequester SMN and concomitantly cause a reduction of SMN levels in the axon, leading to axonal defects. These data provide a functional link between ALS-linked FUS mutations, SMN and neuronal connectivity and support the idea that different motor neuron disorders such as SMA and ALS may be caused, in part, by defects in shared molecular pathways.

Parkinson’s Disease (PD) is a neurodegenerative disease for which there currently is no cure. Aggregation of the pre-synaptic protein α-synuclein (aSN) into oligomers (αSOs) is believed to play a key role in PD pathology, but little is known about αSO formation in vivo and how they induce neurodegeneration. Both the naturally occurring polyunsaturated fatty acid docosahexaenoic acid (DHA) and the lipid peroxidation product 4-hydroxynonenal (HNE), strongly upregulated during ROS conditions, stimulate the formation of αSOs, highlighting a potential role in PD. Yet, insight into αSOs structure and biological effects is still limited as most oligomer preparations studied to date are heterogeneous in composition. Here we have aggregated aSN in the presence of HNE and DHA and purified the αSOs using size exclusion chromatography. Both compounds stimulate formation of spherical αSOs containing anti-parallel β-sheet structure which have the same shape as unmodified αSOs though ca. 2-fold larger. Furthermore, the yield and stabilities of these oligomers are significantly higher than for unmodified aSN. Both modified and unmodified αSOs permeabilize synthetic vesicles, show high co-localisation with glutamatergic synapses and decrease Long Term Potentiation (LTP), in line with the reported synaptotoxic effects of αSOs. We conclude that DHA- and HNE-αSOs are convenient models for pathogenic disease-associated αSOs in PD.

A critical step in Parkinson's disease (PD) is the formation of toxic α‐synuclein oligomers (αSOs). In vitro αSOs are formed by self‐assembly of α‐synuclein at high concentrations, or by the addition of, for example, dopamine, lipids, ethanol, or metal ions. These αSOs are structurally distinct from the unfolded monomer and aggregated β‐sheet fibrils. Nevertheless, the literature reports a wide variety of αSO shapes, sizes, and proposed toxic mechanisms. This heterogeneous character makes it difficult to form a unifying picture. Here, we present an overview of the different αSO species made in vitro, providing a tool for better comparison of different protocols and the ensuing αSOs, and emphasizing the striking versatility in the appearance and properties of these critical species. We also summarize what is known of the biological activities of different αSOs. Despite a large and increasing level of insight into αSO effects in vitro, we still lack strong insight into the structures and sizes of αSO species formed in vivo. Once this is established, it may be possible to generate more uniform protocols that could stimulate further efforts to develop viable PD biomarker assays and therapies.

While it is generally accepted that a-synuclein oligomers (aSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes aSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative aSO binding partners in rat primary neurons using DHA-stabilized human aSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the aSOsbinding partners. We identify in total 29 proteins associated with DHA-aSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium-potassium pump (Na + /K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified aSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between aSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic aSOs. AbbreviationsCo-IP, co-immunoprecipitation; LC-MS/MS, liquid chromatography coupled to mass spectrometric analysis and tandem mass spectrometry; NHS, N-hydroxysuccinimide; NMDA, N-methyl-D-aspartate; PD, Parkinson's disease; sulfo-LC-SDA, sulfo-linker carbon-succinimidyl-diazirine; aSOs, a-synuclein oligomers.Proteomic identification of aSN oligomer ligands F. van Diggelen et al.