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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

HGS expression in Hgs+/+ and Hgstn/tn tissues.(A) qPCR analysis of Hgs mRNA expression in 4-week-old Hgs+/+ tissues. Transcript level is expressed relative to Hgs level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old Hgs+/+ (wt) and Hgstn/tn (tn) mice. β-actin was used as a loading control. (C) qPCR analysis of Hgs levels from the brains of Hgs+/+ mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in Hgs+/+ (wt) and Hgstn/tn (tn) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in Hgs+/+ (wt) and Hgstn/tn (tn) mice expressed as percent of E15 Hgs+/+ levels. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. A one way anova with a Geisser-Greenhouse adjustment demonstrated a significant difference between time points. (F) Quantitation of HGS expression in Hgstn/tn mice expressed as a percent of Hgs+/+ controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.
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pgen.1005290.g002: HGS expression in Hgs+/+ and Hgstn/tn tissues.(A) qPCR analysis of Hgs mRNA expression in 4-week-old Hgs+/+ tissues. Transcript level is expressed relative to Hgs level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old Hgs+/+ (wt) and Hgstn/tn (tn) mice. β-actin was used as a loading control. (C) qPCR analysis of Hgs levels from the brains of Hgs+/+ mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in Hgs+/+ (wt) and Hgstn/tn (tn) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in Hgs+/+ (wt) and Hgstn/tn (tn) mice expressed as percent of E15 Hgs+/+ levels. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. A one way anova with a Geisser-Greenhouse adjustment demonstrated a significant difference between time points. (F) Quantitation of HGS expression in Hgstn/tn mice expressed as a percent of Hgs+/+ controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.

Mentions: To investigate the distribution of Hgs expression in vivo, we performed quantitative polymerase chain reaction (qPCR)on reverse-transcribed RNA isolated from 4-week-old Hgs+/+ wild type mice. Hgs was expressed in all tissues examined, with the highest levels of Hgs detected in the brain and spinal cord (Fig 2A). Consistent with the qPCR data, immunoblot analysis also revealed high levels of HGS in the nervous system of wild type mice (Fig 2B). While the tn mutation resulted in a significant reduction of HGS in the nervous system of the Hgstn/tn mice, it differentially affected HGS levels in non-neuronal tissues (Fig 2B). Examination of the heart, liver, spleen, kidney, adrenal gland, thalamus, testis, and ovaries from the Hgstn/tn mice revealed a striking absence of pathology in these tissues, suggesting that HGS provides an essential function that is unique to the nervous system.


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)

HGS expression in Hgs+/+ and Hgstn/tn tissues.(A) qPCR analysis of Hgs mRNA expression in 4-week-old Hgs+/+ tissues. Transcript level is expressed relative to Hgs level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old Hgs+/+ (wt) and Hgstn/tn (tn) mice. β-actin was used as a loading control. (C) qPCR analysis of Hgs levels from the brains of Hgs+/+ mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in Hgs+/+ (wt) and Hgstn/tn (tn) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in Hgs+/+ (wt) and Hgstn/tn (tn) mice expressed as percent of E15 Hgs+/+ levels. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. A one way anova with a Geisser-Greenhouse adjustment demonstrated a significant difference between time points. (F) Quantitation of HGS expression in Hgstn/tn mice expressed as a percent of Hgs+/+ controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.
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pgen.1005290.g002: HGS expression in Hgs+/+ and Hgstn/tn tissues.(A) qPCR analysis of Hgs mRNA expression in 4-week-old Hgs+/+ tissues. Transcript level is expressed relative to Hgs level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old Hgs+/+ (wt) and Hgstn/tn (tn) mice. β-actin was used as a loading control. (C) qPCR analysis of Hgs levels from the brains of Hgs+/+ mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in Hgs+/+ (wt) and Hgstn/tn (tn) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in Hgs+/+ (wt) and Hgstn/tn (tn) mice expressed as percent of E15 Hgs+/+ levels. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. A one way anova with a Geisser-Greenhouse adjustment demonstrated a significant difference between time points. (F) Quantitation of HGS expression in Hgstn/tn mice expressed as a percent of Hgs+/+ controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.
Mentions: To investigate the distribution of Hgs expression in vivo, we performed quantitative polymerase chain reaction (qPCR)on reverse-transcribed RNA isolated from 4-week-old Hgs+/+ wild type mice. Hgs was expressed in all tissues examined, with the highest levels of Hgs detected in the brain and spinal cord (Fig 2A). Consistent with the qPCR data, immunoblot analysis also revealed high levels of HGS in the nervous system of wild type mice (Fig 2B). While the tn mutation resulted in a significant reduction of HGS in the nervous system of the Hgstn/tn mice, it differentially affected HGS levels in non-neuronal tissues (Fig 2B). Examination of the heart, liver, spleen, kidney, adrenal gland, thalamus, testis, and ovaries from the Hgstn/tn mice revealed a striking absence of pathology in these tissues, suggesting that HGS provides an essential function that is unique to the nervous system.

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