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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

GlyRSP234KY secretion leads to non-cell autonomous build-up at the neuronal membrane. (A–F) L3 larval NMJs were stained under non-permeabilization conditions to assess GlyRSP234KY binding to the neuronal extracellular membrane. (A) HA staining was not observed at the membrane in control flies. (B) Ubiquitous garsP234KY expression, but not (C) garsWT expression, leads to a build-up of GlyRS at the neuronal membrane. (D) Mesodermal and (E) muscle expression of garsP234KY also causes a build-up of GlyRSP234KY on the neuronal membrane. GlyRSP234KY appeared to associate with the whole pre-synapse and axonal regions. (F) Neuronally expressed GlyRSP234KY did not localize to the synapse. (G) Quantification of the HA fluorescence at the synapse. Ten NMJs were analysed per genotype. Scale bars = 10 µm.
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DDV176F4: GlyRSP234KY secretion leads to non-cell autonomous build-up at the neuronal membrane. (A–F) L3 larval NMJs were stained under non-permeabilization conditions to assess GlyRSP234KY binding to the neuronal extracellular membrane. (A) HA staining was not observed at the membrane in control flies. (B) Ubiquitous garsP234KY expression, but not (C) garsWT expression, leads to a build-up of GlyRS at the neuronal membrane. (D) Mesodermal and (E) muscle expression of garsP234KY also causes a build-up of GlyRSP234KY on the neuronal membrane. GlyRSP234KY appeared to associate with the whole pre-synapse and axonal regions. (F) Neuronally expressed GlyRSP234KY did not localize to the synapse. (G) Quantification of the HA fluorescence at the synapse. Ten NMJs were analysed per genotype. Scale bars = 10 µm.

Mentions: As both developmental and degenerative processes appear to affect the mutant gars synapse, we next analysed the localization of GlyRSP234KY. As the canonical function of GlyRS is strictly intracellular, but there appears to be a non-cell autonomous component to pathology in our model, we studied the extracellular localization of GlyRSP234KY using non-permeabilization conditions (without detergent) (Fig. 4). The relative florescence of GlyRS at the synapse was measured and normalized to the background intensity (Fig. 4G). When garsP234KY was ubiquitously expressed, GlyRSP234KY was found to broadly cover the neuromuscular synapse in puncta that were mainly closely associated with the HRP-labelled pre-synaptic membrane (Fig. 4A and B). Signal was not detectable in controls or when garsWT was expressed (Fig. 4C). This was again seen when GlyRSP234KY was driven independently in the mesoderm (Fig. 4D) or in the muscle (Fig. 4E). These experiments suggest that GlyRSP234KY, but not GlyRSWT, is secreted into the extracellular milieu at the NMJ and is able to localize to the outside of the pre-synaptic membrane. In addition, neuron-expressed GlyRSP234KY (elav-GAL4) did not appear at the pre-synapse (Fig. 4F), suggesting that the synaptic build-up of mutant GlyRS is due to a non-cell autonomous mechanism. Ubiquitous, mesodermal and muscle-expressed mutant GlyRS localized to the NMJ when using both UAS-garsP234KY_1 and UAS-garsP234KY_2 constructs (Fig. 4G), whereas muscle-expressed GlyRSWT did not (Supplementary Material, Fig. S1B).Figure 4.


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)

GlyRSP234KY secretion leads to non-cell autonomous build-up at the neuronal membrane. (A–F) L3 larval NMJs were stained under non-permeabilization conditions to assess GlyRSP234KY binding to the neuronal extracellular membrane. (A) HA staining was not observed at the membrane in control flies. (B) Ubiquitous garsP234KY expression, but not (C) garsWT expression, leads to a build-up of GlyRS at the neuronal membrane. (D) Mesodermal and (E) muscle expression of garsP234KY also causes a build-up of GlyRSP234KY on the neuronal membrane. GlyRSP234KY appeared to associate with the whole pre-synapse and axonal regions. (F) Neuronally expressed GlyRSP234KY did not localize to the synapse. (G) Quantification of the HA fluorescence at the synapse. Ten NMJs were analysed per genotype. Scale bars = 10 µm.
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Related In: Results  -  Collection

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DDV176F4: GlyRSP234KY secretion leads to non-cell autonomous build-up at the neuronal membrane. (A–F) L3 larval NMJs were stained under non-permeabilization conditions to assess GlyRSP234KY binding to the neuronal extracellular membrane. (A) HA staining was not observed at the membrane in control flies. (B) Ubiquitous garsP234KY expression, but not (C) garsWT expression, leads to a build-up of GlyRS at the neuronal membrane. (D) Mesodermal and (E) muscle expression of garsP234KY also causes a build-up of GlyRSP234KY on the neuronal membrane. GlyRSP234KY appeared to associate with the whole pre-synapse and axonal regions. (F) Neuronally expressed GlyRSP234KY did not localize to the synapse. (G) Quantification of the HA fluorescence at the synapse. Ten NMJs were analysed per genotype. Scale bars = 10 µm.
Mentions: As both developmental and degenerative processes appear to affect the mutant gars synapse, we next analysed the localization of GlyRSP234KY. As the canonical function of GlyRS is strictly intracellular, but there appears to be a non-cell autonomous component to pathology in our model, we studied the extracellular localization of GlyRSP234KY using non-permeabilization conditions (without detergent) (Fig. 4). The relative florescence of GlyRS at the synapse was measured and normalized to the background intensity (Fig. 4G). When garsP234KY was ubiquitously expressed, GlyRSP234KY was found to broadly cover the neuromuscular synapse in puncta that were mainly closely associated with the HRP-labelled pre-synaptic membrane (Fig. 4A and B). Signal was not detectable in controls or when garsWT was expressed (Fig. 4C). This was again seen when GlyRSP234KY was driven independently in the mesoderm (Fig. 4D) or in the muscle (Fig. 4E). These experiments suggest that GlyRSP234KY, but not GlyRSWT, is secreted into the extracellular milieu at the NMJ and is able to localize to the outside of the pre-synaptic membrane. In addition, neuron-expressed GlyRSP234KY (elav-GAL4) did not appear at the pre-synapse (Fig. 4F), suggesting that the synaptic build-up of mutant GlyRS is due to a non-cell autonomous mechanism. Ubiquitous, mesodermal and muscle-expressed mutant GlyRS localized to the NMJ when using both UAS-garsP234KY_1 and UAS-garsP234KY_2 constructs (Fig. 4G), whereas muscle-expressed GlyRSWT did not (Supplementary Material, Fig. S1B).Figure 4.

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