Periodic Reporting for period 4 - CMTaaRS (Defective protein translation as a pathogenic mechanism of peripheral neuropathy)
Período documentado: 2022-12-01 hasta 2024-05-31
Neurodegenerative diseases are characterized by adult-onset progressive degeneration of specific neuronal populations. The molecular mechanisms underlying neurodegenerative diseases are poorly understood, and as a consequence, there are currently no or only minimally effective (disease-modifying) treatments available. Thus, there is a high unmet medical need, and neurodegenerative diseases are a high burden to patients and their families and result in high costs for society.
This is also the case for Charcot-Marie-Tooth (CMT) peripheral neuropathy, which is caused by selective degeneration of peripheral motor and sensory neurons, leading to progressive muscle weakness and wasting, steppage gait, often foot deformities, and sensory dysfunction. Currently, there is not a single FDA- or EMA-approved drug available for CMT.
The overall goal of this project was to unravel the molecular mechanisms underlying specific genetic forms of CMT, caused by mutations in cytoplasmic tRNA synthetases (CMT-aaRS). tRNA synthetases constitute the largest protein family implicated in CMT, because dominantly inherited (heterozygous) mutations in 8 distinct tRNA synthetase genes all cause CMT. tRNA synthetases are enzymes which link amino acids to their cognate tRNA, thus catalyzing the first step of protein biosynthesis. We had previously shown that expression of CMT-mutant versions tRNA synthetases in Drosophila motor or sensory neurons resulted in a significant reduction of global protein synthesis.
The specific aims of the project were to determine whether translation is also inhibited in CMT-aaRS mouse models, and whether all mutations cause disease through gain-of-toxic-function, or alternatively, whether some mutations act through a dominant-negative mechanism. In addition, the major goal was to unravel the molecular mechanism underlying CMT-aaRS.
This is also the case for Charcot-Marie-Tooth (CMT) peripheral neuropathy, which is caused by selective degeneration of peripheral motor and sensory neurons, leading to progressive muscle weakness and wasting, steppage gait, often foot deformities, and sensory dysfunction. Currently, there is not a single FDA- or EMA-approved drug available for CMT.
The overall goal of this project was to unravel the molecular mechanisms underlying specific genetic forms of CMT, caused by mutations in cytoplasmic tRNA synthetases (CMT-aaRS). tRNA synthetases constitute the largest protein family implicated in CMT, because dominantly inherited (heterozygous) mutations in 8 distinct tRNA synthetase genes all cause CMT. tRNA synthetases are enzymes which link amino acids to their cognate tRNA, thus catalyzing the first step of protein biosynthesis. We had previously shown that expression of CMT-mutant versions tRNA synthetases in Drosophila motor or sensory neurons resulted in a significant reduction of global protein synthesis.
The specific aims of the project were to determine whether translation is also inhibited in CMT-aaRS mouse models, and whether all mutations cause disease through gain-of-toxic-function, or alternatively, whether some mutations act through a dominant-negative mechanism. In addition, the major goal was to unravel the molecular mechanism underlying CMT-aaRS.
We have studied the molecular mechanisms underlying Charcot-Marie-Tooth disease caused by mutations in tRNA synthetases. We have identified the molecular mechanism underlying CMT caused by mutations in glycyl-tRNA synthetase (CMT2D). We could show that CMT-mutant glycyl-tRNA synthetase is still able to bind glycyl-tRNA, but fails to release it. This sequestration of glycyl-tRNA depletes the cellular pool of glycyl-tRNA, leaving insufficient substrate for wild type glycyl-tRNA synthetase (encoded by the wild type gene copy in heterozygous patients). This results in insufficient supply of aminoacylated glycyl-tRNA-Gly to the ribosome and stalling of the ribosome on glycine codons.
This mechanism also suggests that elevating glycyl-tRNA levels may constitute a completely novel therapeutic approach for CMT2D. We could indeed show that transgenic overexpression of glycyl-tRNA in CMT2D mouse models fully prevented peripheral neuropathy at least until the age of 1 year. We are currently evaluating the therapeutic effect of AAV9-mediated viral gene transfer of glycyl-tRNA in CMT2D mouse models, and our unpublished data show that administration of AAV9-glycyl-tRNA to presymptomatic CMT2D mice fully rescues peripheral neuropathy phenotypes. We have patented the use of tRNA overexpression as a therapeutic approach for CMT associated with mutations in tRNA synthetases, and we recently established a spin-off company that aims to develop AAV9-glycyl-tRNA gene therapy for CMT2D patients.
We have further investigated whether a similar mechanism underlies CMT caused by mutations in tyrosyl-tRNA synthetase (DI-CMTC) and histidyl-tRNA synthetase (CMT2W), and whether all CMT-causing mutations in glycyl-tRNA synthetase cause diseases through glycyl-tRNA sequestration, or alternatively, whether some mutations may cause disease through a dominant negative mechanism (leading to a substantial reduction in glycyl-tRNA aminoacylation). The results of these studies are currently being prepared for publication.
We have disseminated the results of the project in published papers (18 thus far, 4 additional ones in the pipeline), invited lectures on conferences, workshops or at universities (>30), and public outreach (press releases, news features on webpages, participation in public outreach events, interviews in newspapers, etc).
This mechanism also suggests that elevating glycyl-tRNA levels may constitute a completely novel therapeutic approach for CMT2D. We could indeed show that transgenic overexpression of glycyl-tRNA in CMT2D mouse models fully prevented peripheral neuropathy at least until the age of 1 year. We are currently evaluating the therapeutic effect of AAV9-mediated viral gene transfer of glycyl-tRNA in CMT2D mouse models, and our unpublished data show that administration of AAV9-glycyl-tRNA to presymptomatic CMT2D mice fully rescues peripheral neuropathy phenotypes. We have patented the use of tRNA overexpression as a therapeutic approach for CMT associated with mutations in tRNA synthetases, and we recently established a spin-off company that aims to develop AAV9-glycyl-tRNA gene therapy for CMT2D patients.
We have further investigated whether a similar mechanism underlies CMT caused by mutations in tyrosyl-tRNA synthetase (DI-CMTC) and histidyl-tRNA synthetase (CMT2W), and whether all CMT-causing mutations in glycyl-tRNA synthetase cause diseases through glycyl-tRNA sequestration, or alternatively, whether some mutations may cause disease through a dominant negative mechanism (leading to a substantial reduction in glycyl-tRNA aminoacylation). The results of these studies are currently being prepared for publication.
We have disseminated the results of the project in published papers (18 thus far, 4 additional ones in the pipeline), invited lectures on conferences, workshops or at universities (>30), and public outreach (press releases, news features on webpages, participation in public outreach events, interviews in newspapers, etc).
The elucidation of the detailed molecular working mechanism underlying inhibition of protein synthesis and the induction of peripheral neuropathy phenotypes in Drosophila and mouse models for CMT caused by mutant tRNA synthetases constitutes a major breakthrough and allows rational drug design for this currently incurable disease. We have established a spin-off company XtRNA Bio, which aims to develop tRNA gene therapy for CMT patients with mutations in tRNA synthetases.