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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

Positional cloning and phenotypic analysis of the tn mice.(A) Image showing reduced size of 4-week-old Hgstn/tn mice relative to wild type HGS+/+ mice. (B) Body mass of 3- to 5-week-old HGS+/+, heterozygous Hgstn/+ and Hgstn/tn mice. n > 6 mice per genotype. A two-way anova was used to find a significant effect of genotype on body mass. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method (C) Kaplan-Meier survival curve of wild type (Hgs+/+) and Hgs-mutant mice. The HgsKO allele does not complement the Hgstn allele. A Mantel-Cox test with p<0.001 demonstrated a significant difference between the survival curves of the Hgstn/tn and HrsKO/tn mice as compared to the HgKO/+Hgstn/+, and Hgs+/+ mice. (D) Meiotic linkage map depicting SNP markers that define the tn critical region. (E) Genomic sequencing of HGS revealed an adenine to guanine change in the Hgstn/tn mice, resulting in a methionine to valine substitution at amino acid 89 of HGS. (F) Schematic of HGS protein structure in eukaryotes, demonstrating the conservation of the methionine residue at position 89 in the VHS domain (orange box). Data are shown as mean ± SE. *p < 0.05 and ***p < 0.001.
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pgen.1005290.g001: Positional cloning and phenotypic analysis of the tn mice.(A) Image showing reduced size of 4-week-old Hgstn/tn mice relative to wild type HGS+/+ mice. (B) Body mass of 3- to 5-week-old HGS+/+, heterozygous Hgstn/+ and Hgstn/tn mice. n > 6 mice per genotype. A two-way anova was used to find a significant effect of genotype on body mass. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method (C) Kaplan-Meier survival curve of wild type (Hgs+/+) and Hgs-mutant mice. The HgsKO allele does not complement the Hgstn allele. A Mantel-Cox test with p<0.001 demonstrated a significant difference between the survival curves of the Hgstn/tn and HrsKO/tn mice as compared to the HgKO/+Hgstn/+, and Hgs+/+ mice. (D) Meiotic linkage map depicting SNP markers that define the tn critical region. (E) Genomic sequencing of HGS revealed an adenine to guanine change in the Hgstn/tn mice, resulting in a methionine to valine substitution at amino acid 89 of HGS. (F) Schematic of HGS protein structure in eukaryotes, demonstrating the conservation of the methionine residue at position 89 in the VHS domain (orange box). Data are shown as mean ± SE. *p < 0.05 and ***p < 0.001.

Mentions: The tn mutation arose spontaneously in a C3H/HeJ inbred mouse line, resulting in a progressive neurodevelopmental disorder that first presents at 3 weeks of age [36]. Homozygous tn mice exhibit reduced growth, ataxia, hypokinesis and premature death at 4 to 5 weeks of age (Fig 1A–1C) [36]. These deficits are thought to be due to dysgenesis of the brainstem and spinal cord [36]. To identify the minimal chromosomal region harboring the tn mutation, a congenic mouse line was generated by backcrossing the tn mutation onto the C57BL/6J genetic background. Analysis of single nucleotide polymorphisms localized the tn mutation to a 2.8 Mb region on distal chromosome 11 (Fig 1D). We then performed transcriptome analysis of brain RNA to identify nucleotide changes in the coding sequences of the genes located within the tn critical region. Sequence analysis of the 87 genes within the tn critical region only revealed a single nucleotide substitution in the Hgs gene. Genomic sequencing of the Hgs gene from the tn mice confirmed an adenine to guanine transition at position 265, which resulted in a methionine to valine substitution at amino acid 89 (M89V) (Fig 1E). The methionine residue at position 89 of HGS is highly conserved throughout eukaryotic phylogeny (Fig 1F).


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)

Positional cloning and phenotypic analysis of the tn mice.(A) Image showing reduced size of 4-week-old Hgstn/tn mice relative to wild type HGS+/+ mice. (B) Body mass of 3- to 5-week-old HGS+/+, heterozygous Hgstn/+ and Hgstn/tn mice. n > 6 mice per genotype. A two-way anova was used to find a significant effect of genotype on body mass. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method (C) Kaplan-Meier survival curve of wild type (Hgs+/+) and Hgs-mutant mice. The HgsKO allele does not complement the Hgstn allele. A Mantel-Cox test with p<0.001 demonstrated a significant difference between the survival curves of the Hgstn/tn and HrsKO/tn mice as compared to the HgKO/+Hgstn/+, and Hgs+/+ mice. (D) Meiotic linkage map depicting SNP markers that define the tn critical region. (E) Genomic sequencing of HGS revealed an adenine to guanine change in the Hgstn/tn mice, resulting in a methionine to valine substitution at amino acid 89 of HGS. (F) Schematic of HGS protein structure in eukaryotes, demonstrating the conservation of the methionine residue at position 89 in the VHS domain (orange box). Data are shown as mean ± SE. *p < 0.05 and ***p < 0.001.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482608&req=5

pgen.1005290.g001: Positional cloning and phenotypic analysis of the tn mice.(A) Image showing reduced size of 4-week-old Hgstn/tn mice relative to wild type HGS+/+ mice. (B) Body mass of 3- to 5-week-old HGS+/+, heterozygous Hgstn/+ and Hgstn/tn mice. n > 6 mice per genotype. A two-way anova was used to find a significant effect of genotype on body mass. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method (C) Kaplan-Meier survival curve of wild type (Hgs+/+) and Hgs-mutant mice. The HgsKO allele does not complement the Hgstn allele. A Mantel-Cox test with p<0.001 demonstrated a significant difference between the survival curves of the Hgstn/tn and HrsKO/tn mice as compared to the HgKO/+Hgstn/+, and Hgs+/+ mice. (D) Meiotic linkage map depicting SNP markers that define the tn critical region. (E) Genomic sequencing of HGS revealed an adenine to guanine change in the Hgstn/tn mice, resulting in a methionine to valine substitution at amino acid 89 of HGS. (F) Schematic of HGS protein structure in eukaryotes, demonstrating the conservation of the methionine residue at position 89 in the VHS domain (orange box). Data are shown as mean ± SE. *p < 0.05 and ***p < 0.001.
Mentions: The tn mutation arose spontaneously in a C3H/HeJ inbred mouse line, resulting in a progressive neurodevelopmental disorder that first presents at 3 weeks of age [36]. Homozygous tn mice exhibit reduced growth, ataxia, hypokinesis and premature death at 4 to 5 weeks of age (Fig 1A–1C) [36]. These deficits are thought to be due to dysgenesis of the brainstem and spinal cord [36]. To identify the minimal chromosomal region harboring the tn mutation, a congenic mouse line was generated by backcrossing the tn mutation onto the C57BL/6J genetic background. Analysis of single nucleotide polymorphisms localized the tn mutation to a 2.8 Mb region on distal chromosome 11 (Fig 1D). We then performed transcriptome analysis of brain RNA to identify nucleotide changes in the coding sequences of the genes located within the tn critical region. Sequence analysis of the 87 genes within the tn critical region only revealed a single nucleotide substitution in the Hgs gene. Genomic sequencing of the Hgs gene from the tn mice confirmed an adenine to guanine transition at position 265, which resulted in a methionine to valine substitution at amino acid 89 (M89V) (Fig 1E). The methionine residue at position 89 of HGS is highly conserved throughout eukaryotic phylogeny (Fig 1F).

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