Limits...
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

Comparison of hippocampal structure and protein expression between Hgstn/tn and Hgs+/+ mice.(A) CA3 hippocampal sections from 4-week-old Hgs+/+ and Hgstn/tn mice were stained for Nissl, myelin basic protein (MBP), glial fibrillary protein (GFAP) and activated caspase 3. Inset is positive control (E13 embryo) for activated caspase-3. Nuclei are stained with DAPI (blue). (B) Representative immunoblot of hippocampal lysates from 4-week-old Hgs+/+ and Hgstn/tn mice. Blots were probed for the ESCRT components HGS, STAM1, and CHMP2B, the receptor tyrosine kinases TrkA and TrkB, and the autophagic markers LC3 and p62. (C) Quantitation of immunoblots from hippocampal lysates. n = 3 mice per genotype. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482608&req=5

pgen.1005290.g003: Comparison of hippocampal structure and protein expression between Hgstn/tn and Hgs+/+ mice.(A) CA3 hippocampal sections from 4-week-old Hgs+/+ and Hgstn/tn mice were stained for Nissl, myelin basic protein (MBP), glial fibrillary protein (GFAP) and activated caspase 3. Inset is positive control (E13 embryo) for activated caspase-3. Nuclei are stained with DAPI (blue). (B) Representative immunoblot of hippocampal lysates from 4-week-old Hgs+/+ and Hgstn/tn mice. Blots were probed for the ESCRT components HGS, STAM1, and CHMP2B, the receptor tyrosine kinases TrkA and TrkB, and the autophagic markers LC3 and p62. (C) Quantitation of immunoblots from hippocampal lysates. n = 3 mice per genotype. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.

Mentions: A previous study of a neuronal-specific knockout of Hgs demonstrated multiple deficits in the hippocampus that included increased ubiquitin staining of CA3 pyramidal cells, increased CA3 pyramidal cell death, and reduced CA3 pyramidal cell numbers [43]. To determine whether the Hgstn/tn mice exhibited any alterations in the number of CA3 pyramidal cells or an increase in cell death, we measured the number of CA3 cells and looked for evidence of increased cell death by performing Nissl and activated caspase-3 staining, respectively, in the hippocampi of wild type and Hgstn/tn mice. Unlike what was reported for the neuronal-specific Hgs knockout mice, we did not detect any change in the number of CA3 pyramidal cells or an increase in cell death markers in the Hgstn/tn hippocampus (Fig 3A). In addition, the levels of hippocampal myelin basic protein were similar in the Hgstn/tn and Hgs+/+ mice. Moreover, while increased glial fibrillary acid protein (GFAP) abundance is often associated with neurodegeneration, there was no increase in GFAP immunoreactivity in the hippocampus of the Hgstn/tn mice compared to wild type controls (Fig 3A). Similar to what we observed in total brain extracts, there was a significant reduction in both HGS and STAM1 levels in the hippocampus of the Hgstn/tn mice. Since mutations in CHMP2B are associated with neurodegeneration in humans, we examined the effect of loss of HGS on CHMP2B levels and found no detectable differences in the hippocampus between the Hgstn/tn or wild type controls. Examination of the levels of the receptor tyrosine kinases TrkA and TrkB, which are putative substrates for HGS, in hippocampal extracts from the Hgstn/tn and Hgs+/+ mice also did not reveal any significant differences in expression (Fig 3B and 3C).


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)

Comparison of hippocampal structure and protein expression between Hgstn/tn and Hgs+/+ mice.(A) CA3 hippocampal sections from 4-week-old Hgs+/+ and Hgstn/tn mice were stained for Nissl, myelin basic protein (MBP), glial fibrillary protein (GFAP) and activated caspase 3. Inset is positive control (E13 embryo) for activated caspase-3. Nuclei are stained with DAPI (blue). (B) Representative immunoblot of hippocampal lysates from 4-week-old Hgs+/+ and Hgstn/tn mice. Blots were probed for the ESCRT components HGS, STAM1, and CHMP2B, the receptor tyrosine kinases TrkA and TrkB, and the autophagic markers LC3 and p62. (C) Quantitation of immunoblots from hippocampal lysates. n = 3 mice per genotype. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005290.g003: Comparison of hippocampal structure and protein expression between Hgstn/tn and Hgs+/+ mice.(A) CA3 hippocampal sections from 4-week-old Hgs+/+ and Hgstn/tn mice were stained for Nissl, myelin basic protein (MBP), glial fibrillary protein (GFAP) and activated caspase 3. Inset is positive control (E13 embryo) for activated caspase-3. Nuclei are stained with DAPI (blue). (B) Representative immunoblot of hippocampal lysates from 4-week-old Hgs+/+ and Hgstn/tn mice. Blots were probed for the ESCRT components HGS, STAM1, and CHMP2B, the receptor tyrosine kinases TrkA and TrkB, and the autophagic markers LC3 and p62. (C) Quantitation of immunoblots from hippocampal lysates. n = 3 mice per genotype. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
Mentions: A previous study of a neuronal-specific knockout of Hgs demonstrated multiple deficits in the hippocampus that included increased ubiquitin staining of CA3 pyramidal cells, increased CA3 pyramidal cell death, and reduced CA3 pyramidal cell numbers [43]. To determine whether the Hgstn/tn mice exhibited any alterations in the number of CA3 pyramidal cells or an increase in cell death, we measured the number of CA3 cells and looked for evidence of increased cell death by performing Nissl and activated caspase-3 staining, respectively, in the hippocampi of wild type and Hgstn/tn mice. Unlike what was reported for the neuronal-specific Hgs knockout mice, we did not detect any change in the number of CA3 pyramidal cells or an increase in cell death markers in the Hgstn/tn hippocampus (Fig 3A). In addition, the levels of hippocampal myelin basic protein were similar in the Hgstn/tn and Hgs+/+ mice. Moreover, while increased glial fibrillary acid protein (GFAP) abundance is often associated with neurodegeneration, there was no increase in GFAP immunoreactivity in the hippocampus of the Hgstn/tn mice compared to wild type controls (Fig 3A). Similar to what we observed in total brain extracts, there was a significant reduction in both HGS and STAM1 levels in the hippocampus of the Hgstn/tn mice. Since mutations in CHMP2B are associated with neurodegeneration in humans, we examined the effect of loss of HGS on CHMP2B levels and found no detectable differences in the hippocampus between the Hgstn/tn or wild type controls. Examination of the levels of the receptor tyrosine kinases TrkA and TrkB, which are putative substrates for HGS, in hippocampal extracts from the Hgstn/tn and Hgs+/+ mice also did not reveal any significant differences in expression (Fig 3B and 3C).

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