Molecular control of local translation in axon development and maintenance

The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons

Genetics of Amyotrophic Lateral Sclerosis

La sclérose latérale amyotrophique (SLA) est la maladie des neurones moteurs la plus fréquente, affectant 4-6 individus par 100,000 habitants à l’échelle mondiale. La maladie se caractérise par une faiblesse et une atrophie musculaire suite à la dégénérescence des neurones du cortex moteur, tronc cérébral et moelle épinière. Les personnes atteintes développent les premiers symptômes à l’âge adulte et la maladie progresse sur une période de trois à cinq ans. Il a été répertorié qu’environ 10% des patients ont une histoire familiale de SLA; 90% des gens affectés le sont donc de façon sporadique. La découverte il y a 19 ans de mutations dans le gène zinc/copper superoxide dismutase (SOD1), présentes dans 15-20% des cas familiaux de SLA et environ 2% du total des individus affectés, a été l’événement déclencheur pour la découverte de variations génétiques responsables de la maladie. La recherche sur la génétique de la SLA a connu une progression rapide ces quatre dernières années avec l’identification de mutations dans de nouveaux gènes. Toutefois, même si certains de ces gènes ont été démontrés comme réellement liés à la maladie, la contribution d’autres gènes demeure incertaine puisque les résultats publiés de ceux-ci n’ont pas, à ce jour, été répliqués. Une portion substantielle de cas reste cependant à être génétiquement expliquée, et aucun traitement à ce jour n’a été démontré comme étant efficace pour remédier, atténuer ou prévenir la maladie. Le but du projet de recherche de doctorat était d’identifier de nouveaux gènes mutés dans la SLA, tout en évaluant la contribution de gènes nouvellement identifiés chez une importante cohorte multiethnique de cas familiaux et sporadiques. Les résultats présentés sont organisés en trois sections différentes. Dans un premier temps, la contribution de mutations présentes dans le gène FUS est évaluée chez les patients familiaux, sporadiques et juvéniles de SLA. Précisément, de nouvelles mutations sont rapportées et la proportion de mutations retrouvées chez les cas familiaux et sporadiques de SLA est évaluée. De plus, une nouvelle mutation est rapportée dans un cas juvénile de SLA; cette étude de cas est discutée. Dans un deuxième temps, de nouvelles avenues génétiques sont explorées concernant le gène SOD1. En effet, une nouvelle mutation complexe est rapportée chez une famille française de SLA. De plus, la possibilité qu’une mutation présente dans un autre gène impliqué dans la SLA ait un impact sur l’épissage du gène SOD1 est évaluée. Finalement, la dernière section explique la contribution de nouveaux gènes candidats chez les patients atteints de SLA. Spécifiquement, le rôle des gènes OPTN, SIGMAR1 et SORT1 dans le phénotype de SLA est évalué. Il est souhaité que nos résultats combinés avec les récents développements en génétique et biologie moléculaire permettent une meilleure compréhension du mécanisme pathologique responsable de cette terrible maladie tout en guidant le déploiement de thérapies suite à l’identification des cibles appropriées.Amyotrophic lateral sclerosis (ALS) is the most common of motor neuron diseases, affecting 4-6 individuals per 100,000 individuals worldwide. ALS is characterized by muscle weakness and atrophy caused by the degeneration of neurons located in the motor cortex, brain stem and spinal cord. This fatal disease generally has an adult onset and progresses over a three to five year period. While 10% of patients affected have a family history of the disease, 90% of cases do not and are considered sporadic. The finding of mutations in the zinc/copper superoxide dismutase gene (SOD1) gene 19 years ago in about 15-20% of familial ALS (FALS) patients and approximately 2% of overall cases developed the interest of identifying rare genetics variants causing the disease. The ALS research field experienced a rapid progression during the last four years as mutations in new genes have been identified. While mutations in some of those new genes have been clearly linked to ALS, the role of others is still questionable and so far has not been positively replicated in other populations. Importantly, a significant portion of cases still need to be genetically explained and, unfortunately, there is still no effective treatment to cure, attenuate or prevent the disease. The aim of this Ph.D research project was to identify new ALS mutated genes while analysing the causative role of other newly identified genes in a large familial and sporadic ALS cohort of different origins. The results presented here are categorized into three different sections. First, the contribution of FUS mutations to familial, sporadic and juvenile ALS is analysed. Specifically, new FUS mutations are reported in ALS cases and the proportions of variants present in the tested familial and sporadic ALS cohorts are assessed. In addition, a new mutation is reported in a juvenile ALS patient, and this interesting case is discussed. Second, new genetic avenues are explored for the SOD1 gene. Precisely, a new and complex SOD1 mutation is reported in a French ALS family. Moreover, the possibility that other ALS mutated genes influence SOD1 splicing events is evaluated. Third, the contribution of new candidate genes is evaluated. Precisely, the contribution of OPTN, SIGMAR1 and SORT1 genes to the ALS phenotype is assessed. Hopefully, our different findings combined with recent developments in genetics and molecular biology will permit a better understanding of the pathological mechanisms involved in the disease and will lead to the identification of the right targets in order to develop appropriate therapeutics for ALS patients

Chapter Genetics of Amyotrophic Lateral Sclerosis

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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

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons.

Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.This work was supported by Wellcome Trust Grants (085314/Z/08/Z and 203249/Z/16/Z) to C.E.H. and (100329/Z/12/Z) to W.A.H., European Research Council Advanced Grant (322817) to C.E.H., Champalimaud Vision Award to C.E.H. and by the Netherlands Organization for Scientific Research (NWO Rubicon 019.161LW.033) to M.K. CFK acknowledges funding from the UK Engineering and Physical Sciences Research Council, EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council, MRC (grants MR/K015850/1 and MR/K02292X/1) and Infinitus (China) Ltd

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

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Molecular control of local translation in axon development and maintenance

The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons

Organelle distribution in neurons: Logistics behind polarized transport

Highly polarized neurons need to carefully regulate the distribution of organelles and other cargoes into their two morphologically and functionally distinct domains, the somatodendritic and axonal compartments, to maintain proper neuron homeostasis. An outstanding question in the field is how organelles reach their correct destination. Long-range transport along microtubules, driven by motors, ensures a fast and controlled availability of organelles in axons and dendrites, but it remains largely unclear what rules govern their transport into the correct compartment. Here, we review the emerging concepts of polarized cargo trafficking in neurons, highlighting the role of microtubule organization, microtubule-associated proteins, and motor proteins and discuss compartment-specific inclusion and exclusion mechanisms as well as the regulation of correct coupling of cargoes to motor proteins