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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

Levels of HGS-interacting proteins and putative substrates in spinal cord extracts of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot and (B) quantitation of the ESCRT-0 proteins HGS and STAM1, the ESCRT-I protein TSG101, the ESCRT-0 interacting proteins EPS15, and the receptor tyrosine kinases TrkB, TrkA and EGFR in the spinal cords of Hgs+/+ and Hgstn/tn mice. β-tubulin was used as a loading control. (C) qPCR analysis of Hgs and Stam1 in the spinal cords of Hgs+/+ and Hgstn/tn mice. Levels are expressed relative to levels found in wild type Hgs+/+ mice. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. (D) Motor neuron counts from lumbar segments 4/5 from Hgs+/+ and Hgstn/tn mice. n = 3 mice per genotype. (E) Immunostaining of Hgs+/+ and Hgstn/tn L4/5 segments with GFAP. Scale bar, 100 μm. Data are shown as mean ± SE. n > 3 mice per genotype. ***p<0.001.
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pgen.1005290.g008: Levels of HGS-interacting proteins and putative substrates in spinal cord extracts of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot and (B) quantitation of the ESCRT-0 proteins HGS and STAM1, the ESCRT-I protein TSG101, the ESCRT-0 interacting proteins EPS15, and the receptor tyrosine kinases TrkB, TrkA and EGFR in the spinal cords of Hgs+/+ and Hgstn/tn mice. β-tubulin was used as a loading control. (C) qPCR analysis of Hgs and Stam1 in the spinal cords of Hgs+/+ and Hgstn/tn mice. Levels are expressed relative to levels found in wild type Hgs+/+ mice. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. (D) Motor neuron counts from lumbar segments 4/5 from Hgs+/+ and Hgstn/tn mice. n = 3 mice per genotype. (E) Immunostaining of Hgs+/+ and Hgstn/tn L4/5 segments with GFAP. Scale bar, 100 μm. Data are shown as mean ± SE. n > 3 mice per genotype. ***p<0.001.

Mentions: Since loss of HGS resulted in a dramatic motor phenotype in the Hgstn/tn mice, but had only a modest effect on peripheral nerve myelination, we examined the effect of reduced HGS expression on the abundance of ESCRT-0 components in the spinal cords of 4-week-old mice (Fig 8A and 8B). Despite the significant increase in Hgs transcript levels in the Hgstn/tn mice (Fig 8C), HGS levels were reduced 70% in the spinal cords of the Hgstn/tn mice compared to controls (Fig 8A and 8B). Similarly, we also observed an 80% reduction in the levels of STAM1 in the spinal cords of the Hgstn/tn mice with no corresponding decrease in Stam1 transcript abundance (Fig 8A–8C). When we examined whether the loss of HGS affected the abundance of other proteins reported to interact with HGS [51–53], there was no difference in the levels of EPS15 or TSG101 in the spinal cords of the Hgstn/tn and Hgs+/+ mice (Fig 8A and 8B). Although HGS appears to influence the abundance of some receptor tyrosine kinases in immortalized cell lines [22,53–58] and TrkB in the sciatic nerves (Fig 7), the levels of EGFR, TrkA and TrkB found in the spinal cords of the Hgstn/tn mice were similar to those observed in the Hgs+/+ control mice (Fig 8A and 8B).


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)

Levels of HGS-interacting proteins and putative substrates in spinal cord extracts of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot and (B) quantitation of the ESCRT-0 proteins HGS and STAM1, the ESCRT-I protein TSG101, the ESCRT-0 interacting proteins EPS15, and the receptor tyrosine kinases TrkB, TrkA and EGFR in the spinal cords of Hgs+/+ and Hgstn/tn mice. β-tubulin was used as a loading control. (C) qPCR analysis of Hgs and Stam1 in the spinal cords of Hgs+/+ and Hgstn/tn mice. Levels are expressed relative to levels found in wild type Hgs+/+ mice. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. (D) Motor neuron counts from lumbar segments 4/5 from Hgs+/+ and Hgstn/tn mice. n = 3 mice per genotype. (E) Immunostaining of Hgs+/+ and Hgstn/tn L4/5 segments with GFAP. Scale bar, 100 μm. Data are shown as mean ± SE. n > 3 mice per genotype. ***p<0.001.
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pgen.1005290.g008: Levels of HGS-interacting proteins and putative substrates in spinal cord extracts of 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Representative immunoblot and (B) quantitation of the ESCRT-0 proteins HGS and STAM1, the ESCRT-I protein TSG101, the ESCRT-0 interacting proteins EPS15, and the receptor tyrosine kinases TrkB, TrkA and EGFR in the spinal cords of Hgs+/+ and Hgstn/tn mice. β-tubulin was used as a loading control. (C) qPCR analysis of Hgs and Stam1 in the spinal cords of Hgs+/+ and Hgstn/tn mice. Levels are expressed relative to levels found in wild type Hgs+/+ mice. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. (D) Motor neuron counts from lumbar segments 4/5 from Hgs+/+ and Hgstn/tn mice. n = 3 mice per genotype. (E) Immunostaining of Hgs+/+ and Hgstn/tn L4/5 segments with GFAP. Scale bar, 100 μm. Data are shown as mean ± SE. n > 3 mice per genotype. ***p<0.001.
Mentions: Since loss of HGS resulted in a dramatic motor phenotype in the Hgstn/tn mice, but had only a modest effect on peripheral nerve myelination, we examined the effect of reduced HGS expression on the abundance of ESCRT-0 components in the spinal cords of 4-week-old mice (Fig 8A and 8B). Despite the significant increase in Hgs transcript levels in the Hgstn/tn mice (Fig 8C), HGS levels were reduced 70% in the spinal cords of the Hgstn/tn mice compared to controls (Fig 8A and 8B). Similarly, we also observed an 80% reduction in the levels of STAM1 in the spinal cords of the Hgstn/tn mice with no corresponding decrease in Stam1 transcript abundance (Fig 8A–8C). When we examined whether the loss of HGS affected the abundance of other proteins reported to interact with HGS [51–53], there was no difference in the levels of EPS15 or TSG101 in the spinal cords of the Hgstn/tn and Hgs+/+ mice (Fig 8A and 8B). Although HGS appears to influence the abundance of some receptor tyrosine kinases in immortalized cell lines [22,53–58] and TrkB in the sciatic nerves (Fig 7), the levels of EGFR, TrkA and TrkB found in the spinal cords of the Hgstn/tn mice were similar to those observed in the Hgs+/+ control mice (Fig 8A and 8B).

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