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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

garsP234KY larvae display NMJ denervation and axonal defects. (A) Representative NMJs from late L3 larvae ubiquitously (1032-GAL4) expressing garsWT and garsP234KY. (B) Representative axonal segment pictures taken of the transverse nerve (TN) over muscles 6 and 7 in abdominal segment A2 in late L3 control, garsWT (1032-GAL4), garsP234KY (1032-GAL4) and garsP234KY mesodermal (how-GAL4) larvae. (C) Quantification of the normalized Brp fluorescence at the NMJ showing ubiquitous, mesodermal and muscle garsP234KY expression leads to reduced Brp signal. (D) Quantification of the number of Brp-positive foci in the TN showing that ubiquitous, mesodermal and muscle garsP234KY expression leads to increased Brp accumulation. For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's/Dunn's multiple comparison test. At least 20 NMJs and axons were analysed per genotype. Scale bars = 10 µm.
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DDV176F3: garsP234KY larvae display NMJ denervation and axonal defects. (A) Representative NMJs from late L3 larvae ubiquitously (1032-GAL4) expressing garsWT and garsP234KY. (B) Representative axonal segment pictures taken of the transverse nerve (TN) over muscles 6 and 7 in abdominal segment A2 in late L3 control, garsWT (1032-GAL4), garsP234KY (1032-GAL4) and garsP234KY mesodermal (how-GAL4) larvae. (C) Quantification of the normalized Brp fluorescence at the NMJ showing ubiquitous, mesodermal and muscle garsP234KY expression leads to reduced Brp signal. (D) Quantification of the number of Brp-positive foci in the TN showing that ubiquitous, mesodermal and muscle garsP234KY expression leads to increased Brp accumulation. For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's/Dunn's multiple comparison test. At least 20 NMJs and axons were analysed per genotype. Scale bars = 10 µm.

Mentions: As the synaptic structure was affected in gars mutant flies, we next looked to see if axonal and synaptic degenerative defects presented at the late L3 larval stage. CMT2D patients display muscle weakness and axonal degeneration (27). In Drosophila, denervation of the synapse can be analysed by detecting the loss or reduction of pre-synaptic antigens such as the active zone protein Brp (28). In ubiquitous, mesodermal and muscle garsP234KY_2-expressing larvae, we found synaptic Brp intensity was reduced compared with control (Fig. 3A and C). This was not seen when garsWT was overexpressed (Fig. 3A and C). It is worth noting that HRP immunoreactivity was reduced at the synapse in ubiquitous and muscle garsP234KY-expressing larvae, suggesting that the defects were due to a degeneration of the pre-synapse and not the mis-regulation of Brp anchoring. In addition to the synaptic denervation, Brp was found to accumulate along the axon (Fig. 3B and D), which is a potential indicator of neurodegeneration (29) or axonal transport defects (30). Together, these findings demonstrate that synaptic and axonal defects can be caused by a non-cell autonomous mechanism.Figure 3.


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)

garsP234KY larvae display NMJ denervation and axonal defects. (A) Representative NMJs from late L3 larvae ubiquitously (1032-GAL4) expressing garsWT and garsP234KY. (B) Representative axonal segment pictures taken of the transverse nerve (TN) over muscles 6 and 7 in abdominal segment A2 in late L3 control, garsWT (1032-GAL4), garsP234KY (1032-GAL4) and garsP234KY mesodermal (how-GAL4) larvae. (C) Quantification of the normalized Brp fluorescence at the NMJ showing ubiquitous, mesodermal and muscle garsP234KY expression leads to reduced Brp signal. (D) Quantification of the number of Brp-positive foci in the TN showing that ubiquitous, mesodermal and muscle garsP234KY expression leads to increased Brp accumulation. For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's/Dunn's multiple comparison test. At least 20 NMJs and axons were analysed per genotype. Scale bars = 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492401&req=5

DDV176F3: garsP234KY larvae display NMJ denervation and axonal defects. (A) Representative NMJs from late L3 larvae ubiquitously (1032-GAL4) expressing garsWT and garsP234KY. (B) Representative axonal segment pictures taken of the transverse nerve (TN) over muscles 6 and 7 in abdominal segment A2 in late L3 control, garsWT (1032-GAL4), garsP234KY (1032-GAL4) and garsP234KY mesodermal (how-GAL4) larvae. (C) Quantification of the normalized Brp fluorescence at the NMJ showing ubiquitous, mesodermal and muscle garsP234KY expression leads to reduced Brp signal. (D) Quantification of the number of Brp-positive foci in the TN showing that ubiquitous, mesodermal and muscle garsP234KY expression leads to increased Brp accumulation. For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's/Dunn's multiple comparison test. At least 20 NMJs and axons were analysed per genotype. Scale bars = 10 µm.
Mentions: As the synaptic structure was affected in gars mutant flies, we next looked to see if axonal and synaptic degenerative defects presented at the late L3 larval stage. CMT2D patients display muscle weakness and axonal degeneration (27). In Drosophila, denervation of the synapse can be analysed by detecting the loss or reduction of pre-synaptic antigens such as the active zone protein Brp (28). In ubiquitous, mesodermal and muscle garsP234KY_2-expressing larvae, we found synaptic Brp intensity was reduced compared with control (Fig. 3A and C). This was not seen when garsWT was overexpressed (Fig. 3A and C). It is worth noting that HRP immunoreactivity was reduced at the synapse in ubiquitous and muscle garsP234KY-expressing larvae, suggesting that the defects were due to a degeneration of the pre-synapse and not the mis-regulation of Brp anchoring. In addition to the synaptic denervation, Brp was found to accumulate along the axon (Fig. 3B and D), which is a potential indicator of neurodegeneration (29) or axonal transport defects (30). Together, these findings demonstrate that synaptic and axonal defects can be caused by a non-cell autonomous mechanism.Figure 3.

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