Distinct effects of ubiquitin overexpression on NMJ structure and motor performance in mice expressing catalytically inactive USP14

Ubiquitin-specific protease 14 (USP14) is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ) structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (axJ) mice and demonstrated that transgenic ubiquitin overexpression partially rescues the axJ neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the axJ deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically-inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the axJ mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway

Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

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

HGS expression in <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> tissues.

<p>(A) qPCR analysis of <i>Hgs</i> mRNA expression in 4-week-old <i>Hgs</i>^<i>+/+</i> tissues. Transcript level is expressed relative to <i>Hgs</i> level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) mice. β-actin was used as a loading control. (C) qPCR analysis of <i>Hgs</i> levels from the brains of <i>Hgs</i>^<i>+/+</i> mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) mice expressed as percent of E15 <i>Hgs</i>^<i>+/+</i> 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 <i>Hgs</i>^<i>tn/tn</i> mice expressed as a percent of <i>Hgs</i>^<i>+/+</i> controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.</p

Positional cloning and phenotypic analysis of the <i>tn</i> mice.

<p>(A) Image showing reduced size of 4-week-old <i>Hgs</i>^<i>tn/tn</i> mice relative to wild type <i>HGS</i>^<i>+/+</i> mice. (B) Body mass of 3- to 5-week-old <i>HGS</i>^<i>+/+</i>, heterozygous <i>Hgs</i>^<i>tn/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. n > 6 mice per genotype. A two-way anova was used to find a significant effect of genotype on body mass. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method (C) Kaplan-Meier survival curve of wild type (<i>Hgs</i>^<i>+/+</i>) and <i>Hgs-</i>mutant mice. The <i>Hgs</i>^<i>KO</i> allele does not complement the <i>Hgs</i>^<i>tn</i> allele. A Mantel-Cox test with p<0.001 demonstrated a significant difference between the survival curves of the <i>Hgs</i>^<i>tn/tn</i> and <i>Hrs</i>^<i>KO/tn</i> mice as compared to the <i>Hg</i>^<i>KO/+</i><i>Hgs</i>^<i>tn/+</i>, and <i>Hgs</i>^<i>+/+</i> mice. (D) Meiotic linkage map depicting SNP markers that define the <i>tn</i> critical region. (E) Genomic sequencing of <i>HGS</i> revealed an adenine to guanine change in the <i>Hgs</i>^<i>tn/tn</i> mice, resulting in a methionine to valine substitution at amino acid 89 of HGS. (F) Schematic of HGS protein structure in eukaryotes, demonstrating the conservation of the methionine residue at position 89 in the VHS domain (orange box). Data are shown as mean ± SE. *p < 0.05 and ***p < 0.001.</p

Alterations in muscles and motor endplates in the <i>Hgs</i>^<i>tn/tn</i> mice.

<p>(A) Wet weights of gastrocnemius muscles from 4 week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. (B) Ratio of gastrocnemius muscle weights to body mass. n > 6 mice per genotype for each time point. (C) Gastrocnemius muscle fiber size measurements for <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. n > 6 mice per genotype. Symbols represent unpaired t-tests. (D) qPCR analysis of <i>AChR-α</i>, <i>AChR-β</i>, <i>AChR-δ</i>, <i>AChR-ε</i>, and <i>AChR-γ</i> mRNAs from the gastrocnemius muscles of 4-week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. n <b>></b> 3 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (E) Motor endplate pathology in the <i>Hgs</i>^<i>tn/tn</i> mice. TA muscle fibers from <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice containing the <i>Thy1</i>-<i>Yfp</i> transgene (green) were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic receptors. The presynaptic axons and nerve terminals are shown in green. Arrowheads mark ultra-terminal sprouting, and curved arrows mark swollen presynaptic terminals. Scale bar, 20 μm. (F) Quantitation of terminal swellings and terminal sprouting from <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. n > 6 mice per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. (G) Histogram of endplate area defined by TRITC-α-bungarotoxin (red) labeling of the postsynaptic AChR in <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of endplate size frequency between <i>Hgs</i>^+/+ and <i>Hgs</i>^tn/tn mice. n > 6 mice per genotype. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.</p

Examination of sciatic nerves from 4-week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice.

<p>(A) Electron micrograph of sciatic nerves from 4-week-old <i>Hgs</i>^<i>tn/tn</i> and <i>Hgs</i>^<i>+/+</i> 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 <i>Hgs</i>^<i>tn/tn</i> mice relative to <i>Hgs</i>^<i>+/+</i> controls. Shaded region represents axonal size distribution from <i>Hgs</i>^<i>+/+</i> mice. An unpaired t-test with a Welch’s correction demonstrated a significant difference in the distribution of axonal size frequency between <i>Hgs</i>^+/+ and <i>Hgs</i>^tn/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 <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> sciatic nerves. Circled region depicts 1.0–2.0 μm diameter axons that are affected in the <i>Hgs</i>^<i>tn/tn</i> sciatic nerves. (G) Representative micrographs of myelin pathology in <i>Hgs</i>^<i>tn/tn</i> nerves demonstrating (1–2) Tomaculous fibers, (3–5) myelin infoldings compared to (6) <i>Hgs</i>^<i>+/+</i>controls. n = 3 mice per genotype. Scale bar, 5 μm. Data are shown as mean ± SE. *p<0.05 and ***p<0.001.</p

Loss of HGS increases the number of endosome-like structures and results in synaptic transmission deficits at the NMJ.

<p>(A) Representative electron micrographs of NMJs in the TA muscle from 4-week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. Arrowheads point to endosomes-like structures. Asterisk marks an MVB in the <i>Hgs</i>^<i>tn/tn</i> presynaptic terminal. No MVBs were observed in <i>Hgs</i>^<i>+/+</i> terminals. Scale bar, 500 μm. (B) Quantitation of endosome-like structures at the motor axon terminals. Symbol represents unpaired t-tests. (C) A 50% reduction in EPC amplitudes was observed in the endplates from 3-week-old <i>Hgs</i>^<i>tn/tn</i> mice (n = 12 endplates from 6 mice) as compared to controls (n = 12 endplates from 5 mice). (D) MEPC amplitudes were reduced in the <i>Hgs</i>^<i>tn/tn</i> mice (n = 19 endplates from 6 mice) to 46% of <i>Hgs</i>^<i>+/+</i> controls (n = 15 endplates from 8 mice). (E) Quantal content was significantly lower in <i>Hgs</i>^<i>tn/tn</i> mice (n = 8 endplates from 6 mice) than in <i>Hgs</i>^<i>+/+</i> controls (n = 12 endplates from 5 mice). (F) Reduced HGS expression results in a 65% reduction in MEPC frequency at the TA muscles of <i>Hgs</i>^<i>tn/tn</i> mice (n = 17 endplates from 6 mice) compared to <i>Hgs</i>^<i>+/+</i>controls (n = 14 endplates from 5 mice). Symbol represents unpaired t-tests **p<0.01 and ***p<0.001.</p

Levels of HGS-interacting proteins and putative substrates in spinal cord extracts of 4-week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice.

<p>(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 <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. β-tubulin was used as a loading control. (C) qPCR analysis of <i>Hgs</i> and <i>Stam1</i> in the spinal cords of <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. Levels are expressed relative to levels found in wild type <i>Hgs</i>^<i>+/+</i> 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 <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> mice. n = 3 mice per genotype. (E) Immunostaining of <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> L4/5 segments with GFAP. Scale bar, 100 μm. Data are shown as mean ± SE. n > 3 mice per genotype. ***p<0.001.</p

Distribution of HGS in sciatic nerves of 4-week-old <i>Hgs</i>^<i>+/+</i>mice.

<p>Top panel, cross sections of sciatic nerves stained with antibodies against neurofilament (NF, green) and HGS (red). Bottom panel, sciatic nerves were stained with antibodies to the Schwann cell marker S100β (green) and HGS (red). Scale bar, 10 μm.</p