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

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

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

Comparison of hippocampal structure and protein expression between <i>Hgs</i>^<i>tn/tn</i> and <i>Hgs</i>^<i>+/+</i> mice.

<p>(A) CA3 hippocampal sections from 4-week-old <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> 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 <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> 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.</p

Gene dosage effects of HGS expression on motor and sensory function in 3- to 4-week-old mice.

<p>(A) Reduced HGS expression results in clawed paws in <i>Hgs</i>^<i>tn/tn</i> mice. Behavioral assays of (B) open field, (C) rotarod, (D) elevated beam, (E) von Frey and (F) forelimb grip strength for <i>Hgs</i>^<i>+/+</i> (black), <i>Hgs</i>^<i>tn/+</i>, <i>Hgs</i>^<i>tn/tn</i> and <i>Hgs</i>^KO/+ mice. n = 6 mice per genotype for all assays except rotarod, where n = 4. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE and n>6 animals per genotype for all assays except rotarod, where n = 4. A two-way anova demonstrated a significant effect of genotype on rotarod and elevated beam performance. Data are shown as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001.</p

Effect of reduced HGS expression on ubiquitin conjugates in the nervous system of <i>Hgs</i>^<i>tn/tn</i> mice.

<p>(A) Representative immunoblot of ubiquitin conjugates from the nervous system of 4-week-old <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) mice. (B) Quantitation of immunoblots. n = 3 per genotype. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. Data are shown as mean ± SE. **p<0.001.</p