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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

Dominant, toxic effects caused by GlyRSP234KY lead to non-cell autonomous longevity and motor defects. (A) GlyRSWT and (B) GlyRSP234KY localization in the central nervous system of larvae ubiquitously (1032-GAL4) expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. (C) GlyRSWT and (D) GlyRSP234KY localization at the NMJ in larvae neuronally expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. Note that GlyRS expressed in neurons does not reach the peripheral synapse. (E and F) GlyRSP234KY toxicity was analysed by expressing garsP234KY ubiquitously (1032-GAL4), in mesoderm (how-GAL4), muscle (MHC-GAL4) or neurons (elav-GAL4) at 20°C (E) and 25°C (F). Transgene expression is lower at 20 than 25°C. Flies ubiquitously expressing garsWT (blue bars) and CD8::GFP (red bars) were used as controls. (G) Mutant garsP234KY flies reach the pupal stage, but fail to eclose and often become trapped in their pupal cases (arrow). The few flies that escape and reach adulthood display wing expansion defects (arrow head). (H) Ubiquitous, mesodermal and muscle expression of garsP234KY caused a significant loss of muscle contractions at 20°C. (I) Ubiquitous, mesodermal, muscle and neuronal driven garsP234KY expression also reduced larval muscle contractions at 25°C. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001 Dunn's multiple comparison test. For survival studies, 25 flies per genotype were scored over four independent experiments. For muscle contraction studies, at least 15 larvae were analysed per genotype. Scale bars = 10 μm.
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DDV176F1: Dominant, toxic effects caused by GlyRSP234KY lead to non-cell autonomous longevity and motor defects. (A) GlyRSWT and (B) GlyRSP234KY localization in the central nervous system of larvae ubiquitously (1032-GAL4) expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. (C) GlyRSWT and (D) GlyRSP234KY localization at the NMJ in larvae neuronally expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. Note that GlyRS expressed in neurons does not reach the peripheral synapse. (E and F) GlyRSP234KY toxicity was analysed by expressing garsP234KY ubiquitously (1032-GAL4), in mesoderm (how-GAL4), muscle (MHC-GAL4) or neurons (elav-GAL4) at 20°C (E) and 25°C (F). Transgene expression is lower at 20 than 25°C. Flies ubiquitously expressing garsWT (blue bars) and CD8::GFP (red bars) were used as controls. (G) Mutant garsP234KY flies reach the pupal stage, but fail to eclose and often become trapped in their pupal cases (arrow). The few flies that escape and reach adulthood display wing expansion defects (arrow head). (H) Ubiquitous, mesodermal and muscle expression of garsP234KY caused a significant loss of muscle contractions at 20°C. (I) Ubiquitous, mesodermal, muscle and neuronal driven garsP234KY expression also reduced larval muscle contractions at 25°C. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001 Dunn's multiple comparison test. For survival studies, 25 flies per genotype were scored over four independent experiments. For muscle contraction studies, at least 15 larvae were analysed per genotype. Scale bars = 10 μm.

Mentions: To mimic the dominant GARS mutations that cause CMT2D, and to better understand the selective neuronal pathology seen in the disease, we expressed the cytoplasmic isoform of wild-type (UAS-garsWT, which produces GlyRSWT) and mutant (UAS-garsP234KY, which produces GlyRSP234KY) Drosophila gars transgenes using the UAS-GAL4 system (18). Two different constructs were used for both the wild-type (UAS-garsWT_1 and UAS-garsWT_2) and the mutant (UAS-garsP234KY_1 and UAS-garsP234KY_2) transgenes, with UAS-garsWT_1 and UAS-garsP234KY_1 being the same as those used by Ermanoska et al. (18), and UAS-garsWT_2 and UAS-garsP234KY_2 being novel to this work. UAS-garsWT_1 and UAS-garsP234KY_1 were previously shown to be expressed at equivalent levels and both proteins localized to the cytoplasm within the neuronal populations studied (18). Here, we first analysed the localization of the GlyRS proteins expressed from our new UAS-garsWT_2 (Fig. 1A) and UAS-garsP234KY_2 (Fig. 1B) constructs, when driven ubiquitously (1032-GAL4). Both GlyRSWT and GlyRSP234KY localized in a similar cytoplasmic diffuse pattern in the cell bodies of larval central nervous system neurons (Fig. 1A and B). Both proteins were also observed in the axonal bundles that contain afferent motor and sensory neurons ensheathed in glia (Fig. 1A and B), as well as the neuropil region of axons and dendrites, which was labelled with bruchpilot (Brp, Fig. 1A and B). We also found analogous localization patterns when UAS-garsWT_1 and UAS-garsP234KY_1 were used (data not shown), as previously described (18). As GlyRS was observed in the axonal bundles, we expressed both GlyRSWT and GlyRSP234KY using a neuronal-specific driver (elav-GAL4) to determine whether GlyRS reaches the most distal region of the motor neuron, the NMJ (Fig. 1C and D). Both GlyRSWT and GlyRSP234KY did not become enriched at the larval NMJ, suggesting that neuronally expressed GlyRS does not reach the peripheral synapses of the neuromuscular system.Figure 1.


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)

Dominant, toxic effects caused by GlyRSP234KY lead to non-cell autonomous longevity and motor defects. (A) GlyRSWT and (B) GlyRSP234KY localization in the central nervous system of larvae ubiquitously (1032-GAL4) expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. (C) GlyRSWT and (D) GlyRSP234KY localization at the NMJ in larvae neuronally expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. Note that GlyRS expressed in neurons does not reach the peripheral synapse. (E and F) GlyRSP234KY toxicity was analysed by expressing garsP234KY ubiquitously (1032-GAL4), in mesoderm (how-GAL4), muscle (MHC-GAL4) or neurons (elav-GAL4) at 20°C (E) and 25°C (F). Transgene expression is lower at 20 than 25°C. Flies ubiquitously expressing garsWT (blue bars) and CD8::GFP (red bars) were used as controls. (G) Mutant garsP234KY flies reach the pupal stage, but fail to eclose and often become trapped in their pupal cases (arrow). The few flies that escape and reach adulthood display wing expansion defects (arrow head). (H) Ubiquitous, mesodermal and muscle expression of garsP234KY caused a significant loss of muscle contractions at 20°C. (I) Ubiquitous, mesodermal, muscle and neuronal driven garsP234KY expression also reduced larval muscle contractions at 25°C. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001 Dunn's multiple comparison test. For survival studies, 25 flies per genotype were scored over four independent experiments. For muscle contraction studies, at least 15 larvae were analysed per genotype. Scale bars = 10 μm.
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Related In: Results  -  Collection

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DDV176F1: Dominant, toxic effects caused by GlyRSP234KY lead to non-cell autonomous longevity and motor defects. (A) GlyRSWT and (B) GlyRSP234KY localization in the central nervous system of larvae ubiquitously (1032-GAL4) expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. (C) GlyRSWT and (D) GlyRSP234KY localization at the NMJ in larvae neuronally expressing UAS-garsWT_2 and UAS-garsP234KY_2, respectively. Note that GlyRS expressed in neurons does not reach the peripheral synapse. (E and F) GlyRSP234KY toxicity was analysed by expressing garsP234KY ubiquitously (1032-GAL4), in mesoderm (how-GAL4), muscle (MHC-GAL4) or neurons (elav-GAL4) at 20°C (E) and 25°C (F). Transgene expression is lower at 20 than 25°C. Flies ubiquitously expressing garsWT (blue bars) and CD8::GFP (red bars) were used as controls. (G) Mutant garsP234KY flies reach the pupal stage, but fail to eclose and often become trapped in their pupal cases (arrow). The few flies that escape and reach adulthood display wing expansion defects (arrow head). (H) Ubiquitous, mesodermal and muscle expression of garsP234KY caused a significant loss of muscle contractions at 20°C. (I) Ubiquitous, mesodermal, muscle and neuronal driven garsP234KY expression also reduced larval muscle contractions at 25°C. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001 Dunn's multiple comparison test. For survival studies, 25 flies per genotype were scored over four independent experiments. For muscle contraction studies, at least 15 larvae were analysed per genotype. Scale bars = 10 μm.
Mentions: To mimic the dominant GARS mutations that cause CMT2D, and to better understand the selective neuronal pathology seen in the disease, we expressed the cytoplasmic isoform of wild-type (UAS-garsWT, which produces GlyRSWT) and mutant (UAS-garsP234KY, which produces GlyRSP234KY) Drosophila gars transgenes using the UAS-GAL4 system (18). Two different constructs were used for both the wild-type (UAS-garsWT_1 and UAS-garsWT_2) and the mutant (UAS-garsP234KY_1 and UAS-garsP234KY_2) transgenes, with UAS-garsWT_1 and UAS-garsP234KY_1 being the same as those used by Ermanoska et al. (18), and UAS-garsWT_2 and UAS-garsP234KY_2 being novel to this work. UAS-garsWT_1 and UAS-garsP234KY_1 were previously shown to be expressed at equivalent levels and both proteins localized to the cytoplasm within the neuronal populations studied (18). Here, we first analysed the localization of the GlyRS proteins expressed from our new UAS-garsWT_2 (Fig. 1A) and UAS-garsP234KY_2 (Fig. 1B) constructs, when driven ubiquitously (1032-GAL4). Both GlyRSWT and GlyRSP234KY localized in a similar cytoplasmic diffuse pattern in the cell bodies of larval central nervous system neurons (Fig. 1A and B). Both proteins were also observed in the axonal bundles that contain afferent motor and sensory neurons ensheathed in glia (Fig. 1A and B), as well as the neuropil region of axons and dendrites, which was labelled with bruchpilot (Brp, Fig. 1A and B). We also found analogous localization patterns when UAS-garsWT_1 and UAS-garsP234KY_1 were used (data not shown), as previously described (18). As GlyRS was observed in the axonal bundles, we expressed both GlyRSWT and GlyRSP234KY using a neuronal-specific driver (elav-GAL4) to determine whether GlyRS reaches the most distal region of the motor neuron, the NMJ (Fig. 1C and D). Both GlyRSWT and GlyRSP234KY did not become enriched at the larval NMJ, suggesting that neuronally expressed GlyRS does not reach the peripheral synapses of the neuromuscular system.Figure 1.

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