tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.

C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A.

A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.

Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity.

Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.

Molecular pathogenesis of peripheral neuropathies: insights from Drosophila models.

Peripheral neuropathies are characterized by degeneration of peripheral motor, sensory and/or autonomic axons, leading to progressive distal muscle weakness, sensory deficits and/or autonomic dysfunction. Acquired peripheral neuropathies, e.g., as a side effect of chemotherapy, are distinguished from inherited peripheral neuropathies (IPNs). Drosophila models for chemotherapy-induced peripheral neuropathy and several IPNs have provided novel insight into the molecular mechanisms underlying axonal degeneration. Forward genetic screens have predictive value for discovery of human IPN genes, and the pathogenicity of novel mutations in known IPN genes can be evaluated in Drosophila. Future screens for genes and compounds that modify Drosophila IPN phenotypes promise to make valuable contributions to unraveling the molecular pathogenesis and identification of therapeutic targets for these incurable diseases.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila.

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

Cell-selective labelling of proteomes in Drosophila melanogaster.

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRS(LtoG)) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through 'click chemistry'. To test these methods for applicability in vivo, we expressed MetRS(LtoG) cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms.

Oxidative stress-induced posttranslational modifications of alpha-synuclein: specific modification of alpha-synuclein by 4-hydroxy-2-nonenal increases dopaminergic toxicity.

Aggregation and neurotoxicity of misfolded alpha-synuclein (αSyn) are crucial mechanisms for progressive dopaminergic neurodegeneration associated with Parkinson's disease (PD). Posttranslational modifications (PTMs) of αSyn caused by oxidative stress, including modification by 4-hydroxy-2-nonenal (HNE-αSyn), nitration (n-αSyn), and oxidation (o-αSyn), have been implicated to promote oligomerization of αSyn. However, it is yet unclear if these PTMs lead to different types of oligomeric intermediates. Moreover, little is known about which PTM-derived αSyn species exerts toxicity to dopaminergic cells. In this study, we directly compared aggregation characteristics of HNE-αSyn, n-αSyn, and o-αSyn. Generally, all of them promoted αSyn oligomerization. Particularly, HNE-αSyn and n-αSyn were more prone to forming oligomers than unmodified αSyn. Moreover, these PTMs prevented the formation of amyloid-like fibrils, although HNE-αSyn and o-αSyn were able to generate protofibrillar structures. The cellular effects associated with distinct PTMs were studied by exposing modified αSyn to dopaminergic Lund human mesencephalic (LUHMES) neurons. The cellular toxicity of HNE-αSyn was significantly higher than other PTM species. Furthermore, we tested the toxicity of HNE-αSyn in dopaminergic LUHMES cells and other cell types with low tyrosine hydroxylase (TH) expression, and additionally analyzed the loss of TH-immunoreactive cells in HNE-αSyn-treated LUHMES cells. We observed a selective toxicity of HNE-αSyn to neurons with higher TH expression. Further mechanistic studies showed that HNE-modification apparently increased the interaction of extracellular αSyn with neurons. Moreover, exposure of differentiated LUHMES cells to HNE-αSyn triggered the production of intracellular reactive oxygen species, preceding neuronal cell death. Antioxidant treatment effectively protected cells from the damage triggered by HNE-αSyn. Our findings suggest a specific pathological effect of HNE-αSyn on dopaminergic neurons.

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes.

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H(2)O(2)/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30-100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.