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Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

Watson JA, Bhattacharyya BJ, Vaden JH, Wilson JA, Icyuz M, Howard AD, Phillips E, DeSilva TM, Siegal GP, Bean AJ, King GD, Phillips SE, Miller RJ, Wilson SM - PLoS Genet. (2015)

Bottom Line: These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ.These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction.Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham, Evelyn F. McKnight Brain Institute, Civitan International Research Center, Birmingham, Alabama, United States of America.

ABSTRACT
Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

No MeSH data available.


Related in: MedlinePlus

Alterations in muscles and motor endplates in the Hgstn/tn mice.(A) Wet weights of gastrocnemius muscles from 4 week-old Hgs+/+ and Hgstn/tn mice. (B) Ratio of gastrocnemius muscle weights to body mass. n > 6 mice per genotype for each time point. (C) Gastrocnemius muscle fiber size measurements for Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests. (D) qPCR analysis of AChR-α, AChR-β, AChR-δ, AChR-ε, and AChR-γ mRNAs from the gastrocnemius muscles of 4-week-old Hgs+/+ and Hgstn/tn mice. n > 3 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (E) Motor endplate pathology in the Hgstn/tn mice. TA muscle fibers from Hgs+/+ and Hgstn/tn mice containing the Thy1-Yfp transgene (green) were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic receptors. The presynaptic axons and nerve terminals are shown in green. Arrowheads mark ultra-terminal sprouting, and curved arrows mark swollen presynaptic terminals. Scale bar, 20 μm. (F) Quantitation of terminal swellings and terminal sprouting from Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (G) Histogram of endplate area defined by TRITC-α-bungarotoxin (red) labeling of the postsynaptic AChR in Hgs+/+ and Hgstn/tn mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of endplate size frequency between Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.
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pgen.1005290.g009: Alterations in muscles and motor endplates in the Hgstn/tn mice.(A) Wet weights of gastrocnemius muscles from 4 week-old Hgs+/+ and Hgstn/tn mice. (B) Ratio of gastrocnemius muscle weights to body mass. n > 6 mice per genotype for each time point. (C) Gastrocnemius muscle fiber size measurements for Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests. (D) qPCR analysis of AChR-α, AChR-β, AChR-δ, AChR-ε, and AChR-γ mRNAs from the gastrocnemius muscles of 4-week-old Hgs+/+ and Hgstn/tn mice. n > 3 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (E) Motor endplate pathology in the Hgstn/tn mice. TA muscle fibers from Hgs+/+ and Hgstn/tn mice containing the Thy1-Yfp transgene (green) were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic receptors. The presynaptic axons and nerve terminals are shown in green. Arrowheads mark ultra-terminal sprouting, and curved arrows mark swollen presynaptic terminals. Scale bar, 20 μm. (F) Quantitation of terminal swellings and terminal sprouting from Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (G) Histogram of endplate area defined by TRITC-α-bungarotoxin (red) labeling of the postsynaptic AChR in Hgs+/+ and Hgstn/tn mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of endplate size frequency between Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.

Mentions: The Hgstn/tn mice exhibit several signs of neuromuscular disease, including muscle weakness and decreased motor performance. When we compared the muscle mass between the Hgstn/tn mice and the Hgs+/+ controls, we detected a significant difference in gastrocnemius weights at 4 weeks of age (Fig 9A and 9B). While we did not detect angular muscle fibers or centrally located nuclei in the muscle sections of the Hgstn/tn mice, which are common features of motor neuron disease, there was a 32% reduction in muscle fiber size in the Hgstn/tn mice (Fig 9C). These changes were consistent with decreased motor neuron input onto the muscle fibers. Since acetylcholine receptor (AChR) abundance is inversely correlated with motor neuron input onto muscle fibers, we performed qPCR on gastrocnemius muscle RNA isolated from 4-week-old wild type and Hgstn/tn mice. Both the embryonic and adult acetylcholine receptor subunit mRNAs were significantly increased in the gastrocnemius muscles of the Hgstn/tn mice (Fig 9D).


Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

Watson JA, Bhattacharyya BJ, Vaden JH, Wilson JA, Icyuz M, Howard AD, Phillips E, DeSilva TM, Siegal GP, Bean AJ, King GD, Phillips SE, Miller RJ, Wilson SM - PLoS Genet. (2015)

Alterations in muscles and motor endplates in the Hgstn/tn mice.(A) Wet weights of gastrocnemius muscles from 4 week-old Hgs+/+ and Hgstn/tn mice. (B) Ratio of gastrocnemius muscle weights to body mass. n > 6 mice per genotype for each time point. (C) Gastrocnemius muscle fiber size measurements for Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests. (D) qPCR analysis of AChR-α, AChR-β, AChR-δ, AChR-ε, and AChR-γ mRNAs from the gastrocnemius muscles of 4-week-old Hgs+/+ and Hgstn/tn mice. n > 3 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (E) Motor endplate pathology in the Hgstn/tn mice. TA muscle fibers from Hgs+/+ and Hgstn/tn mice containing the Thy1-Yfp transgene (green) were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic receptors. The presynaptic axons and nerve terminals are shown in green. Arrowheads mark ultra-terminal sprouting, and curved arrows mark swollen presynaptic terminals. Scale bar, 20 μm. (F) Quantitation of terminal swellings and terminal sprouting from Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (G) Histogram of endplate area defined by TRITC-α-bungarotoxin (red) labeling of the postsynaptic AChR in Hgs+/+ and Hgstn/tn mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of endplate size frequency between Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.
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Related In: Results  -  Collection

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Show All Figures
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pgen.1005290.g009: Alterations in muscles and motor endplates in the Hgstn/tn mice.(A) Wet weights of gastrocnemius muscles from 4 week-old Hgs+/+ and Hgstn/tn mice. (B) Ratio of gastrocnemius muscle weights to body mass. n > 6 mice per genotype for each time point. (C) Gastrocnemius muscle fiber size measurements for Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests. (D) qPCR analysis of AChR-α, AChR-β, AChR-δ, AChR-ε, and AChR-γ mRNAs from the gastrocnemius muscles of 4-week-old Hgs+/+ and Hgstn/tn mice. n > 3 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (E) Motor endplate pathology in the Hgstn/tn mice. TA muscle fibers from Hgs+/+ and Hgstn/tn mice containing the Thy1-Yfp transgene (green) were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic receptors. The presynaptic axons and nerve terminals are shown in green. Arrowheads mark ultra-terminal sprouting, and curved arrows mark swollen presynaptic terminals. Scale bar, 20 μm. (F) Quantitation of terminal swellings and terminal sprouting from Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (G) Histogram of endplate area defined by TRITC-α-bungarotoxin (red) labeling of the postsynaptic AChR in Hgs+/+ and Hgstn/tn mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of endplate size frequency between Hgs+/+ and Hgstn/tn mice. n > 6 mice per genotype. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.
Mentions: The Hgstn/tn mice exhibit several signs of neuromuscular disease, including muscle weakness and decreased motor performance. When we compared the muscle mass between the Hgstn/tn mice and the Hgs+/+ controls, we detected a significant difference in gastrocnemius weights at 4 weeks of age (Fig 9A and 9B). While we did not detect angular muscle fibers or centrally located nuclei in the muscle sections of the Hgstn/tn mice, which are common features of motor neuron disease, there was a 32% reduction in muscle fiber size in the Hgstn/tn mice (Fig 9C). These changes were consistent with decreased motor neuron input onto the muscle fibers. Since acetylcholine receptor (AChR) abundance is inversely correlated with motor neuron input onto muscle fibers, we performed qPCR on gastrocnemius muscle RNA isolated from 4-week-old wild type and Hgstn/tn mice. Both the embryonic and adult acetylcholine receptor subunit mRNAs were significantly increased in the gastrocnemius muscles of the Hgstn/tn mice (Fig 9D).

Bottom Line: These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ.These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction.Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham, Evelyn F. McKnight Brain Institute, Civitan International Research Center, Birmingham, Alabama, United States of America.

ABSTRACT
Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

No MeSH data available.


Related in: MedlinePlus