The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker’s organ through the activity of the Ca2+-activated Cl--channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 (Tmem16a) in mice disrupts Ca2+ waves within Kölliker’s organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker’s organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A

GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options

In brains of aged knockout mice lysosomal compartments show abnormal densities and lysosomal enzyme activities are increased.

<p>(A,B) Density gradient analyses of Lamp1-positive membrane isolations from 20-day-old wild-type and <i>Zfyve26</i> knockout mice with 2 different protocols revealed an increased presence of Lamp1-positive membrane compartments with higher density in <i>Zfyve26</i> knockout material compared to wild-type. (C) A density shift was also observed at 16 months of age (same fractionation protocol as displayed in A). (D) No shift was detected for the AP5M1/μ5 subunit. (E) Quantitative Western blot analysis of Lamp1, Cathepsin D (CtsDm: mature; CtsDp: precursor), or EEA1 in 1.000 g supernatants from brain lysates of 16-month-old mice. Only Cathepsin D levels were significantly increased (n = 5; Student's t-test; *: p<0.05). (F,G) In brain lysates of <i>Zfyve26</i> knockout mice both β-hexosaminidase (F) and β-galactosidase (G) activity was increased at 16 months, but not at 2 months of age (n = 3; 2-way ANOVA; *: p<0.01).</p

Large autofluorescent particles in knockout tissues are Lamp1-positive.

<p>Confocal microscopy of cerebellar sections of 10-month-old mice. (A,B,C,D) Maximum intensity projections of all channels analyzed. (A–B′″) Autofluorescent deposits (green) in Purkinje cells barely co-localized with EEA1 (red). (C–D′″) Autofluorescent deposits in knockout tissues were Lamp1-positive (clone CD107a). Scale bars: 10 µm.</p

Targeted disruption of the murine <i>Zfyve26</i> gene.

<p>(A) Genomic structure of the <i>Zfyve26 gene</i> (top) and the targeted <i>Zfyve26</i> locus (middle). The dotted line indicates the extent of the targeting construct. A neomycin cassette (Neo) flanked by frt sites (black boxes) and a loxP-site (black triangle) was inserted into intron 15. A second loxP-site together with a <i>Bam</i>HI site was introduced into intron 14. Correctly targeted ES cell clones were selected for the generation of chimeric mice. <i>Zfyve26</i> knockout mice were established by breeding chimeric mice to a cre-deleter mouse strain to obtain constitutive <i>Zfyve26</i> knockout mice. (B) Northern blot analysis of total brain RNA from wild-type (WT), <i>Zfyve26</i> heterozygous (HET), and <i>Zfyve26</i> knockout (KO) animals. <i>Gapdh</i> served as loading control. (C,D) Western blot analysis with affinity purified antibodies against the N-terminus (C) or the C-terminus (D) of Zfyve26 detected a 285 kD Zfyve26 polypeptide in brain extracts from wild-type but not from knockout mice. Calnexin served as a loading control. (E) Spatacsin levels, an interaction partner of Zfyve26, are reduced in brain lysates of <i>Zfyve26</i> knockout mice. Calnexin was used as a loading control. (F) <i>Spatacsin</i> is not regulated on the transcriptional level in <i>Zfyve26</i> knockout mice. <i>Gapdh</i> was used as a loading control.</p

Zfyve26 is highly expressed in neuronal cells.

<p>(A) According to the Western blot analysis (80 µg protein per lane) using a novel anti-C-terminus antibody Zfyve26 is expressed in different tissues. (B,C) <i>In situ</i> hybridization of sagittal sections of a brain from a 2-month-old wild-type mouse with a <i>Zfyve26</i> sense-control probe (B) or the <i>Zfyve26</i>-specific antisense probe (C). Higher magnification of labeled areas in (C): (C′) olfactory bulb, (C″) motor cortex, (C′″) hippocampus, (C″″) cerebellar cortex (Purkinje cells are indicated by arrows). (D,D′) <i>In situ</i> hybridization of transversal spinal cord sections. Lower motoneurons in the anterior horn of the spinal cord are labeled as well. Scale bars: 500 µm (B,C), 50 µm (C′–C′″,D), 100 µm (C″″,D′). GCL: granule cell layer, GL: glomeruli, MC: mitral cells, PCL: Purkinje cell layer, PG: periglomerular cells, ML: molecular layer.</p

Zfyve26 is associated with endolysosomal membranes.

<p>(A–C) A ZFYVE26-GFP fusion protein expressed in 3T3 cells was associated with vesicular structures positive for the early endosomal marker protein EEA1. (D–F) There was only a partial association between ZFYVE26-GFP and the late endosomal/lysosomal marker protein Lamp1. (G–I) The vesicular staining was absent in cells transfected with the ZFYVE26-GFP variant harboring the point mutation His1834Ala in the FYVE-domain. (J–L) Pretreatment with wortmannin, an inhibitor of phosphatidylinositol-3-kinases, interfered with the vesicular staining of the FYVE-domain proteins ZFYVE26 and EEA1. Scale bars: 15 µm. (M) Subcellular fractionation of brain homogenates of WT and <i>Zfyve26</i> knockout mice followed by immunoblotting for Zfyve26, its interaction partner AP5M1/μ5, and the marker proteins EEA1 and Lamp1 showed that both Zfyve26 and the AP5 complex were found in the light membrane fraction. Heavy membranes fractions enriched for Lamp1 lacked EEA1, AP5M1/μ5, and Zfyve26-reactive polypeptides.</p

Disruption of <i>Zfyve26</i> causes severe neuron loss in the motor cortex and cerebellum.

<p>(A–B) The brain was smaller in 16-month-old <i>Zfyve26</i> knockout compared to wild-type mice. Scale bars: 2 mm. (C) Progressive reduction of brain weight in <i>Zfyve26</i> knockout mice (n = 4; 2-way ANOVA; **: p<0.001). (D,E) Astrogliosis and loss of NeuN-positive neuronal cells in the motor cortex of 16-month-old <i>Zfyve26</i> knockout mice. Hoechst-33258 (blue; nuclei), GFAP (green; astrocyte marker), and NeuN (red; neuronal marker) staining of the motor cortex at 16 months of age from wild-type (D) and knockout (E) mice. Individual cortical layers are labeled (I–VI). Scale bars: 100 µm. (F) Quantification of NeuN-positive cells per layer revealed a significant reduction of neurons from layers V–VI of the motor cortex in 16-month-old <i>Zfyve26</i> knockout mice (Student's t-test; **: p<0.001). (G–H) Cerebellar sections stained for GFAP (green), Calbindin (red, Purkinje cell marker), and Hoechst-33258 (blue) revealed a severe loss of Purkinje cells in 16-month-old knockout mice. Scale bars: 100 µm. GCL: granule cell layer, PCL: Purkinje cell layer, ML: molecular layer. (I) In knockout mice Purkinje cells were drastically reduced at 16 months (2-way ANOVA; ***: p<0.0001), but not at 2 months of age. (J–K) Semithin sections of the lumbar corticospinal tract illustrates the reduction in the number of large diameter axons in 16-month-old knockout mice. Scale bars: 20 µm. (L) Transmission electron microscopy of a degenerating axon in a <i>Zfyve26</i> knockout mouse. Scale bar: 1 µm. (M,N) Delayed outgrowth of Tau-positive axons of cultured motoneurons isolated from knockout (KO) embryos compared to wild-type (WT) mice. (Student's t-test; **: p<0.001). Scale bar in M: 40 µm. (O) The number of axonal branches in cultured motoneurons did not differ between genotypes. Error bars represent SEM. (Student's t-test; n.s.: not significant).</p