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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

Examination of sciatic nerves from 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Electron micrograph of sciatic nerves from 4-week-old Hgstn/tn and Hgs+/+ mice. Scale bar, 2 μm. Arrowheads indicate hypermyelinated fibers, curved arrows indicated disorganized myelin and arrows indicate demyelination. (B) Quantitation of axon density in myelinated and unmyelinated nerves. (C) Quantitation of average myelinated and unmyelinated axon diameters. (D) Histogram of frequency of axon diameters demonstrating an increase in large diameter myelinated axons in the sciatic nerves of Hgstn/tn mice relative to Hgs+/+ controls. Shaded region represents axonal size distribution from Hgs+/+ mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of axonal size frequency between Hgs+/+ and Hgstn/tn mice. (E) Quantitation of the ratio of axon diameter to total fiber thickness (G-ratio). Symbols represent unpaired t-tests. (F) Relationship between myelin thickness and axon diameter in Hgs+/+ and Hgstn/tn sciatic nerves. Circled region depicts 1.0–2.0 μm diameter axons that are affected in the Hgstn/tn sciatic nerves. (G) Representative micrographs of myelin pathology in Hgstn/tn nerves demonstrating (1–2) Tomaculous fibers, (3–5) myelin infoldings compared to (6) Hgs+/+controls. n = 3 mice per genotype. Scale bar, 5 μm. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
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pgen.1005290.g005: Examination of sciatic nerves from 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Electron micrograph of sciatic nerves from 4-week-old Hgstn/tn and Hgs+/+ mice. Scale bar, 2 μm. Arrowheads indicate hypermyelinated fibers, curved arrows indicated disorganized myelin and arrows indicate demyelination. (B) Quantitation of axon density in myelinated and unmyelinated nerves. (C) Quantitation of average myelinated and unmyelinated axon diameters. (D) Histogram of frequency of axon diameters demonstrating an increase in large diameter myelinated axons in the sciatic nerves of Hgstn/tn mice relative to Hgs+/+ controls. Shaded region represents axonal size distribution from Hgs+/+ mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of axonal size frequency between Hgs+/+ and Hgstn/tn mice. (E) Quantitation of the ratio of axon diameter to total fiber thickness (G-ratio). Symbols represent unpaired t-tests. (F) Relationship between myelin thickness and axon diameter in Hgs+/+ and Hgstn/tn sciatic nerves. Circled region depicts 1.0–2.0 μm diameter axons that are affected in the Hgstn/tn sciatic nerves. (G) Representative micrographs of myelin pathology in Hgstn/tn nerves demonstrating (1–2) Tomaculous fibers, (3–5) myelin infoldings compared to (6) Hgs+/+controls. n = 3 mice per genotype. Scale bar, 5 μm. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.

Mentions: Alterations in axon number or peripheral nerve myelination are common features of inherited peripheral neuropathies and are thought to be important contributors to motor and sensory dysfunction. To examine whether loss of HGS expression results in axonal loss, demyelination, or dysmyelination that could contribute to the behavioral deficits in the Hgstn/tn mice, we compared the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice by transmission electron microscopy. Although no change in the density of myelinated and unmyelinated axons was found in the Hgstn/tn mice (Fig 5A and 5B), there was a significant increase in the diameter of myelinated axons and a significant decrease in the diameter of unmyelinated axons in the 4-week-old Hgstn/tn mice as compared to controls (Fig 5C). This increase in the average axonal diameter of myelinated fibers was attributed to a shift in distribution towards a greater number of large diameter axons in the Hgstn/tn mice (Fig 5D). Morphometric analysis of myelin structure also revealed a small but significant decrease in the G-ratio (the ratio of the inner axonal diameter to the total fiber diameter) of sciatic nerves from the Hgstn/tn mice (Fig 5E). This decrease was attributed to a 22% increase in the myelin thickness of small-diameter axons that are between 1.0 to 2.0 μm in diameter (Fig 5F, circled region).


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)

Examination of sciatic nerves from 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Electron micrograph of sciatic nerves from 4-week-old Hgstn/tn and Hgs+/+ mice. Scale bar, 2 μm. Arrowheads indicate hypermyelinated fibers, curved arrows indicated disorganized myelin and arrows indicate demyelination. (B) Quantitation of axon density in myelinated and unmyelinated nerves. (C) Quantitation of average myelinated and unmyelinated axon diameters. (D) Histogram of frequency of axon diameters demonstrating an increase in large diameter myelinated axons in the sciatic nerves of Hgstn/tn mice relative to Hgs+/+ controls. Shaded region represents axonal size distribution from Hgs+/+ mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of axonal size frequency between Hgs+/+ and Hgstn/tn mice. (E) Quantitation of the ratio of axon diameter to total fiber thickness (G-ratio). Symbols represent unpaired t-tests. (F) Relationship between myelin thickness and axon diameter in Hgs+/+ and Hgstn/tn sciatic nerves. Circled region depicts 1.0–2.0 μm diameter axons that are affected in the Hgstn/tn sciatic nerves. (G) Representative micrographs of myelin pathology in Hgstn/tn nerves demonstrating (1–2) Tomaculous fibers, (3–5) myelin infoldings compared to (6) Hgs+/+controls. n = 3 mice per genotype. Scale bar, 5 μm. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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pgen.1005290.g005: Examination of sciatic nerves from 4-week-old Hgs+/+ and Hgstn/tn mice.(A) Electron micrograph of sciatic nerves from 4-week-old Hgstn/tn and Hgs+/+ mice. Scale bar, 2 μm. Arrowheads indicate hypermyelinated fibers, curved arrows indicated disorganized myelin and arrows indicate demyelination. (B) Quantitation of axon density in myelinated and unmyelinated nerves. (C) Quantitation of average myelinated and unmyelinated axon diameters. (D) Histogram of frequency of axon diameters demonstrating an increase in large diameter myelinated axons in the sciatic nerves of Hgstn/tn mice relative to Hgs+/+ controls. Shaded region represents axonal size distribution from Hgs+/+ mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of axonal size frequency between Hgs+/+ and Hgstn/tn mice. (E) Quantitation of the ratio of axon diameter to total fiber thickness (G-ratio). Symbols represent unpaired t-tests. (F) Relationship between myelin thickness and axon diameter in Hgs+/+ and Hgstn/tn sciatic nerves. Circled region depicts 1.0–2.0 μm diameter axons that are affected in the Hgstn/tn sciatic nerves. (G) Representative micrographs of myelin pathology in Hgstn/tn nerves demonstrating (1–2) Tomaculous fibers, (3–5) myelin infoldings compared to (6) Hgs+/+controls. n = 3 mice per genotype. Scale bar, 5 μm. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.
Mentions: Alterations in axon number or peripheral nerve myelination are common features of inherited peripheral neuropathies and are thought to be important contributors to motor and sensory dysfunction. To examine whether loss of HGS expression results in axonal loss, demyelination, or dysmyelination that could contribute to the behavioral deficits in the Hgstn/tn mice, we compared the sciatic nerves of 4-week-old Hgs+/+ and Hgstn/tn mice by transmission electron microscopy. Although no change in the density of myelinated and unmyelinated axons was found in the Hgstn/tn mice (Fig 5A and 5B), there was a significant increase in the diameter of myelinated axons and a significant decrease in the diameter of unmyelinated axons in the 4-week-old Hgstn/tn mice as compared to controls (Fig 5C). This increase in the average axonal diameter of myelinated fibers was attributed to a shift in distribution towards a greater number of large diameter axons in the Hgstn/tn mice (Fig 5D). Morphometric analysis of myelin structure also revealed a small but significant decrease in the G-ratio (the ratio of the inner axonal diameter to the total fiber diameter) of sciatic nerves from the Hgstn/tn mice (Fig 5E). This decrease was attributed to a 22% increase in the myelin thickness of small-diameter axons that are between 1.0 to 2.0 μm in diameter (Fig 5F, circled region).

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