Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

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

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

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 conf

GSK-3β-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3β (GSK-3β) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein–dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3β is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3β or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3β knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3β and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3β may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization

Axin localizes to the centrosome and is involved in microtubule nucleation

Axin is known to have an important role in the degradation of β-catenin in the Wnt pathway. Here, we reveal a new function of Axin at the centrosome. Axin was localized to the centrosome in various cell lines and formed a complex with γ-tubulin. Knockdown of Axin reduced the localization of γ-tubulin and γ-tubulin complex protein 2—components of the γ-tubulin ring complex—to the centrosome and the centrosomal microtubule nucleation activity after treatment with nocodazole. These phenotypes could not be rescued by the reduction in the levels of β-catenin. Although the expression of Axin rescued these phenotypes in Axin-knockdown cells, overexpression of Axin2, which is highly homologous to Axin, could not. Axin2 was also localized to the centrosome, but it did not form a complex with γ-tubulin. These results suggest that Axin, but not Axin2, is involved in microtubule nucleation by forming a complex with γ-tubulin at the centrosome

ALS-associated mutations in FUS disrupt the axonal distribution and function of SMN

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 pathway