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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

Analysis of ESCRT and RTK expression in the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot of ESCRT expression in sciatic nerve extracts. β-tubulin was used as a loading control. (B) Quantitation of immunoblots of ESCRT expression in sciatic nerve extracts. (C) qPCR analysis of ESCRT-0 components in the sciatic nerve. (D) Representative immunoblot of TrkB.FL and TrkB.T1, EGFR and ERBB2 in sciatic nerves. β-tubulin was used as a loading control. (E) Quantitation of receptor tyrosine kinases in the sciatic nerve. (F) qPCR analysis of TrkB and BDNF in the sciatic nerve. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. n > 3 mice per genotype. **p<0.01 and ***p<0.001.
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pgen.1005290.g007: Analysis of ESCRT and RTK expression in the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot of ESCRT expression in sciatic nerve extracts. β-tubulin was used as a loading control. (B) Quantitation of immunoblots of ESCRT expression in sciatic nerve extracts. (C) qPCR analysis of ESCRT-0 components in the sciatic nerve. (D) Representative immunoblot of TrkB.FL and TrkB.T1, EGFR and ERBB2 in sciatic nerves. β-tubulin was used as a loading control. (E) Quantitation of receptor tyrosine kinases in the sciatic nerve. (F) qPCR analysis of TrkB and BDNF in the sciatic nerve. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. n > 3 mice per genotype. **p<0.01 and ***p<0.001.

Mentions: In cell culture models, the stability of STAM1 appears to be dependent upon HGS expression in a transcript-independent manner [16,50]. However, this relationship between ESCRT-0 components has not been investigated in the nervous system. We therefore examined the effect of the tn mutation on the expression of ESCRT components and their putative substrates in the sciatic nerves of the Hgstn/tn mice. While the HGS levels were reduced by 50% in the sciatic nerves of 4-week-old Hgstn/tn mice compared to controls, there were no significant differences in the levels of STAM1, tumor susceptibility protein 101 (TSG101) or epidermal growth factor receptor substrate 15 (EPS15) in the sciatic nerve extracts from the Hgstn/tn and Hgs+/+ mice (Fig 7A–7C). Analysis of receptor tyrosine kinases (RTKs) sorted by the ESCRT pathway showed that loss of HGS resulted in a significant reduction in both the full length (TrkB.FL) and truncated (TrkB.T1) isoforms of TrkB in the sciatic nerves of the Hgstn/tn mice that was not associated with a corresponding reduction in TrkB mRNA levels (Fig 7D–7F). In contrast, reduction of HGS did not affect the level of either the epidermal-growth factor receptor (EGFR) or the receptor tyrosine-protein kinase erbB-2 (ERBB2) (Fig 7D and 7E).


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)

Analysis of ESCRT and RTK expression in the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot of ESCRT expression in sciatic nerve extracts. β-tubulin was used as a loading control. (B) Quantitation of immunoblots of ESCRT expression in sciatic nerve extracts. (C) qPCR analysis of ESCRT-0 components in the sciatic nerve. (D) Representative immunoblot of TrkB.FL and TrkB.T1, EGFR and ERBB2 in sciatic nerves. β-tubulin was used as a loading control. (E) Quantitation of receptor tyrosine kinases in the sciatic nerve. (F) qPCR analysis of TrkB and BDNF in the sciatic nerve. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. n > 3 mice per genotype. **p<0.01 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005290.g007: Analysis of ESCRT and RTK expression in the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot of ESCRT expression in sciatic nerve extracts. β-tubulin was used as a loading control. (B) Quantitation of immunoblots of ESCRT expression in sciatic nerve extracts. (C) qPCR analysis of ESCRT-0 components in the sciatic nerve. (D) Representative immunoblot of TrkB.FL and TrkB.T1, EGFR and ERBB2 in sciatic nerves. β-tubulin was used as a loading control. (E) Quantitation of receptor tyrosine kinases in the sciatic nerve. (F) qPCR analysis of TrkB and BDNF in the sciatic nerve. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. n > 3 mice per genotype. **p<0.01 and ***p<0.001.
Mentions: In cell culture models, the stability of STAM1 appears to be dependent upon HGS expression in a transcript-independent manner [16,50]. However, this relationship between ESCRT-0 components has not been investigated in the nervous system. We therefore examined the effect of the tn mutation on the expression of ESCRT components and their putative substrates in the sciatic nerves of the Hgstn/tn mice. While the HGS levels were reduced by 50% in the sciatic nerves of 4-week-old Hgstn/tn mice compared to controls, there were no significant differences in the levels of STAM1, tumor susceptibility protein 101 (TSG101) or epidermal growth factor receptor substrate 15 (EPS15) in the sciatic nerve extracts from the Hgstn/tn and Hgs+/+ mice (Fig 7A–7C). Analysis of receptor tyrosine kinases (RTKs) sorted by the ESCRT pathway showed that loss of HGS resulted in a significant reduction in both the full length (TrkB.FL) and truncated (TrkB.T1) isoforms of TrkB in the sciatic nerves of the Hgstn/tn mice that was not associated with a corresponding reduction in TrkB mRNA levels (Fig 7D–7F). In contrast, reduction of HGS did not affect the level of either the epidermal-growth factor receptor (EGFR) or the receptor tyrosine-protein kinase erbB-2 (ERBB2) (Fig 7D and 7E).

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