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Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology.

Grice SJ, Sleigh JN, Motley WW, Liu JL, Burgess RW, Talbot K, Cader MZ - Hum. Mol. Genet. (2015)

Bottom Line: Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology.This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability.Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.

View Article: PubMed Central - PubMed

Affiliation: MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.

No MeSH data available.


Related in: MedlinePlus

GlyRS toxicity and synapse binding, but not secretion, are dependent on the WHEP domain. (A) Removal of the WHEP domain abrogated the pathology associated with both ubiquitous and muscle expression of mutant gars in vivo; garsΔWHEP-P234KY expression had no effect on larval viability. (B) GlyRSΔWHEP-P234KY did not associate with the neuronal membrane (using non-permeabilization conditions). (C) To determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, eGFP-tagged GARSWT and GARSP234KY with or without the WHEP domain were expressed in human kidney (HEK293) and muscle-like (TE671) cells. Deletion of the WHEP domain did not affect secretion of wild-type or mutant GlyRS. The localization of all GlyRS proteins was similar in both cell lines and with each protein tag (data not shown). Scale bars = 10 µm.
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DDV176F5: GlyRS toxicity and synapse binding, but not secretion, are dependent on the WHEP domain. (A) Removal of the WHEP domain abrogated the pathology associated with both ubiquitous and muscle expression of mutant gars in vivo; garsΔWHEP-P234KY expression had no effect on larval viability. (B) GlyRSΔWHEP-P234KY did not associate with the neuronal membrane (using non-permeabilization conditions). (C) To determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, eGFP-tagged GARSWT and GARSP234KY with or without the WHEP domain were expressed in human kidney (HEK293) and muscle-like (TE671) cells. Deletion of the WHEP domain did not affect secretion of wild-type or mutant GlyRS. The localization of all GlyRS proteins was similar in both cell lines and with each protein tag (data not shown). Scale bars = 10 µm.

Mentions: GlyRS was recently shown to be secreted by macrophages and to be present in human and mouse serum, serving as a defence against ERK-activated tumour formation (31). Several other ARS proteins are also known to be secreted (32). The capacity of mesoderm- and muscle-expressed GlyRSP234KY, through secretion, to exert non-cell autonomous effects on the nervous system appears to be an integral component to the pathology we have observed. A candidate determinant for GlyRS secretion is the N-terminal WHEP domain, which currently has no known function in GlyRS and is dispensable for aminoacylation activity (20). We therefore investigated the importance of the WHEP domain for GlyRS toxicity by generating a WHEP-deletion fly. Intriguingly, removal of the WHEP domain from GlyRSP234KY (GlyRSΔWHEP-P234KY) abrogated the fly viability (Fig. 5A) and NMJ defects (data not shown) caused by GlyRSP234KY (Fig. 5A). Furthermore, build-up of GlyRSΔWHEP-P234KY was not found at the NMJ, suggesting that synaptic mutant GlyRS accumulation is integral to pathology (Fig. 5B). To show that our transgenes were successfully expressed and to assess protein localization, we used UAS ‘flip-out’ clones expressing UAS-garsP234KY and UAS-garsΔWHEP-P234KY. Both GlyRSP234KY and GlyRSΔWHEP-P234KY were expressed and localized in the cytoplasm of disc clones (Supplementary Material, Fig. S3A), indicating that deletion of the WHEP domain is unlikely to affect protein stability. This is corroborated by previous observations that deletion of the WHEP domain does not affect the canonical function of GlyRS and does not prevent the crystalization of the truncated protein (20). Finally, to determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, we expressed GARSWT and GARSP234KY with and without the WHEP domain in human kidney (HEK293) and muscle-like (TE671) cells. Contrary to our starting hypothesis that the WHEP domain may be a determinant of secretion, we found that its deletion did not affect secretion of eGFP- or V5-tagged versions of the protein (Fig. 5C, Supplementary Material, Fig. S4A). To confirm that the secretion of GlyRS proteins was not simply a product of the overexpression system, we demonstrated that eGFP-tagged survival motor neuron (Smn) protein is not secreted under similar conditions in both HEK293 (Supplementary Material, Fig. S4B) and TE671 cells (data not shown). The higher expression of Smn-eGFP than GlyRS-eGFP makes this result all the more convincing. Smn was chosen as a control, because, like GlyRS, it is expressed both in the nucleus and cytoplasm (33), it has been identified in axonal projections (10,34), and its dysfunction is associated with a disorder that affects the lower motor neurons (spinal muscular atrophy) (34,35). The similar subcellular localization patterns of eGFP-tagged GlyRS and Smn were confirmed by immunofluorescence (Supplementary Material, Fig. S4C). Altogether, this work indicates that the GlyRS WHEP domain, although not required for secretion, nevertheless is integral to pre-synaptic accumulation of muscle-expressed mutant GlyRS.Figure 5.


Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology.

Grice SJ, Sleigh JN, Motley WW, Liu JL, Burgess RW, Talbot K, Cader MZ - Hum. Mol. Genet. (2015)

GlyRS toxicity and synapse binding, but not secretion, are dependent on the WHEP domain. (A) Removal of the WHEP domain abrogated the pathology associated with both ubiquitous and muscle expression of mutant gars in vivo; garsΔWHEP-P234KY expression had no effect on larval viability. (B) GlyRSΔWHEP-P234KY did not associate with the neuronal membrane (using non-permeabilization conditions). (C) To determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, eGFP-tagged GARSWT and GARSP234KY with or without the WHEP domain were expressed in human kidney (HEK293) and muscle-like (TE671) cells. Deletion of the WHEP domain did not affect secretion of wild-type or mutant GlyRS. The localization of all GlyRS proteins was similar in both cell lines and with each protein tag (data not shown). Scale bars = 10 µm.
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Related In: Results  -  Collection

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Show All Figures
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DDV176F5: GlyRS toxicity and synapse binding, but not secretion, are dependent on the WHEP domain. (A) Removal of the WHEP domain abrogated the pathology associated with both ubiquitous and muscle expression of mutant gars in vivo; garsΔWHEP-P234KY expression had no effect on larval viability. (B) GlyRSΔWHEP-P234KY did not associate with the neuronal membrane (using non-permeabilization conditions). (C) To determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, eGFP-tagged GARSWT and GARSP234KY with or without the WHEP domain were expressed in human kidney (HEK293) and muscle-like (TE671) cells. Deletion of the WHEP domain did not affect secretion of wild-type or mutant GlyRS. The localization of all GlyRS proteins was similar in both cell lines and with each protein tag (data not shown). Scale bars = 10 µm.
Mentions: GlyRS was recently shown to be secreted by macrophages and to be present in human and mouse serum, serving as a defence against ERK-activated tumour formation (31). Several other ARS proteins are also known to be secreted (32). The capacity of mesoderm- and muscle-expressed GlyRSP234KY, through secretion, to exert non-cell autonomous effects on the nervous system appears to be an integral component to the pathology we have observed. A candidate determinant for GlyRS secretion is the N-terminal WHEP domain, which currently has no known function in GlyRS and is dispensable for aminoacylation activity (20). We therefore investigated the importance of the WHEP domain for GlyRS toxicity by generating a WHEP-deletion fly. Intriguingly, removal of the WHEP domain from GlyRSP234KY (GlyRSΔWHEP-P234KY) abrogated the fly viability (Fig. 5A) and NMJ defects (data not shown) caused by GlyRSP234KY (Fig. 5A). Furthermore, build-up of GlyRSΔWHEP-P234KY was not found at the NMJ, suggesting that synaptic mutant GlyRS accumulation is integral to pathology (Fig. 5B). To show that our transgenes were successfully expressed and to assess protein localization, we used UAS ‘flip-out’ clones expressing UAS-garsP234KY and UAS-garsΔWHEP-P234KY. Both GlyRSP234KY and GlyRSΔWHEP-P234KY were expressed and localized in the cytoplasm of disc clones (Supplementary Material, Fig. S3A), indicating that deletion of the WHEP domain is unlikely to affect protein stability. This is corroborated by previous observations that deletion of the WHEP domain does not affect the canonical function of GlyRS and does not prevent the crystalization of the truncated protein (20). Finally, to determine whether WHEP deletion restricts GlyRS secretion and is the principal cause of the abolished mutant pathology, we expressed GARSWT and GARSP234KY with and without the WHEP domain in human kidney (HEK293) and muscle-like (TE671) cells. Contrary to our starting hypothesis that the WHEP domain may be a determinant of secretion, we found that its deletion did not affect secretion of eGFP- or V5-tagged versions of the protein (Fig. 5C, Supplementary Material, Fig. S4A). To confirm that the secretion of GlyRS proteins was not simply a product of the overexpression system, we demonstrated that eGFP-tagged survival motor neuron (Smn) protein is not secreted under similar conditions in both HEK293 (Supplementary Material, Fig. S4B) and TE671 cells (data not shown). The higher expression of Smn-eGFP than GlyRS-eGFP makes this result all the more convincing. Smn was chosen as a control, because, like GlyRS, it is expressed both in the nucleus and cytoplasm (33), it has been identified in axonal projections (10,34), and its dysfunction is associated with a disorder that affects the lower motor neurons (spinal muscular atrophy) (34,35). The similar subcellular localization patterns of eGFP-tagged GlyRS and Smn were confirmed by immunofluorescence (Supplementary Material, Fig. S4C). Altogether, this work indicates that the GlyRS WHEP domain, although not required for secretion, nevertheless is integral to pre-synaptic accumulation of muscle-expressed mutant GlyRS.Figure 5.

Bottom Line: Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology.This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability.Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.

View Article: PubMed Central - PubMed

Affiliation: MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.

No MeSH data available.


Related in: MedlinePlus