Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing

Endothelial FAK is required for tumour angiogenesis

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies

ABR thresholds and SEM assessment suggest a local function of Spns2 in the inner ear.

<p>ABR thresholds (means +/−SD) are shown for homozygotes (red), heterozygotes (blue) and wildtypes (green), aged 7–14 weeks. Mice homozygous for the <i>Spns2^tm1a</i> allele displayed elevated ABR thresholds and degeneration of hair cells (<b><i>A</i></b><i> left</i>, <i>4 wks and </i><b><i>B</i></b>). By crossing with mice expressing Flp recombinase to excise the inserted cassette, <i>Spns2^tm1c/tm1c</i> mice were produced, which had normal ABR thresholds and normal hair cell morphology (<b><i>A</i></b><i> middle</i>, <i>8 wks and </i><b><i>C</i></b>). Then <i>Spns2^tm1c/tm1c</i> were crossed with <i>Sox10-Cre</i> mice to produce <i>Spns2^tm1d/tm1d</i>;Sox10-Cre mice which showed no response up to 95 dB SPL and hair cell degeneration with bulges and holes in the reticular lamina (<b><i>A</i></b><i> right</i>, <i>4 wks and </i><b><i>D</i></b>). SEM images are taken from the middle turn (40–70%) of the cochlea. Scale bar: 10 µm in <b><i>B,C,D</i></b>.</p

ABR thresholds of mice with <i>Spns2</i> conditionally inactivated in different tissues.

<p>ABR thresholds of individual mice are shown. All Cre driver lines showed normal thresholds (left column, in black. n = number of wildtype; number of mice carrying Cre). Most control littermates had normal responses (middle column: heterozygotes in blue; wildtypes in green). Homozygous <i>Spns2^tm1d</i> mutants (red) carrying the relevant Cre alleles are shown in the right column. <i>Spns2^tm1d</i> homozygotes carrying the Sox10-Cre allele had raised thresholds (top right), but the other four Cre lines had normal thresholds. The bottom row shows equivalent threshold data for the <i>Spns2^tm1c</i> allele and the Flp recombinase line used to generate this allele, again showing normal thresholds. There were three exceptions of individuals with raised thresholds (one heterozygote each in Pf4-Cre cross and Tie1-Cre cross, one homozygote in Tie1-Cre cross), which we believe probably carry an independent mutation causing the impairment (subject to ongoing positional cloning study).</p

Eye defects in <i>Spns2^tm1a/tm1a</i> mice.

<p><b><i>A–C</i></b>, Phenotype screening was performed on 15 week old <i>Spns2^tm1a/tm1a</i> mice and identified buphthalmos, corneal opacity with vascularization and possible ulceration (<b><i>B</i></b>) and corneal opacity with vascularisation and polyp, thick discharge, elongated pupil which did not fully dilate with tropicamide (<b><i>C</i></b>). Albino mice were selected to achieve clearer demonstration, and a wildtype is shown in (<b><i>A</i></b>). <b><i>D</i></b>, Pupil-optic nerve section of a 15 week old wildtype eye. <b><i>E</i></b>, The <i>Spns2^tm1a/tm1a</i> mice showed a grossly abnormal eye with corneal opacity, vascularization, collapsed anterior chamber, small cataractous lens, and focal retinal degeneration. c = cornea, ac = anterior chamber, pc = posterior chamber, on = optic nerve. Scale bar = 450 µm. <b><i>F,G</i></b>,<b><i>H,I</i></b> Analysis of retinal vasculature was performed on retinal wholemounts from P10 pups (<b><i>F,G</i></b>) and 8 week old adult mice(<b><i>H,I</i></b>). Retinal vasculature was stained with Isolectin B4 (green) to visualise the endothelium and Proteoglycan NG2 (red) to visualise pericytes. Whereas arteries (a) appeared morphologically normal, veins (v) appeared thinner in <i>Spns2^tm1a/tm1a</i> (<b><i>I</i></b>) than in <i>Spns2^+/tm1a</i> (<b><i>H</i></b>) and had an irregular caliber with regions of narrowing (arrows). Although the retina has three capillary plexi only the primary plexus is shown at both P10 and 8 weeks as this is the first plexus to form and mature. Scalebars: 50 µm. <b><i>J</i></b>, Branch point analysis was performed on P10 retinal vasculature (<b><i>F,G</i></b>) to determine whether retinal vasculature showed any developmental abnormalities in vascular patterning. No significant difference was detected between <i>Spns2^tm1a/tm1a</i> (red) and <i>Spns2^+/tm1a</i> (blue) mice in the central retina (mature vessels) or the periphery, where the vessels are still developing at P10, in either the arteries or veins (Mann-Whitney U test for central branch points, p = 0.69; t-test for peripheral branch points, arterial p = 0.899, venous p = 0.996).</p

The hearing loss of <i>Spns2^tm1a/tm1a</i>, <i>Spns2^tm1b/tm1b</i> and <i>Spns2^tm1d/tm1d</i>; <i>Sox10-Cre</i> mice showed a similar pattern of progression.

<p>ABR thresholds of individual homozygotes (red), heterozygotes (blue) and wildtypes (green) are shown at 2, 3 and 14 or 4–10 weeks old. The control mice had immature thresholds at 2 weeks old and continued to mature to normal hearing levels at 3 weeks old. <i>Spns2^tm1a/tm1a</i> and <i>Spns2^tm1d/tm1d</i>; <i>Sox10-Cre</i> mice displayed progressive hearing loss from 2 to 3 weeks old. The red line in the middle bottom panel represents a control mouse homozygous for <i>Spns2^tm1c</i> but without carrying <i>Sox10-Cre</i>, and thus had no conditional knockout of <i>Spns2</i> in the inner ear and normal hearing.</p

Normal strial integrity and normal permeability of strial capillaries to BSA-FITC.

<p><b><i>A,B</i></b>, Endolymphatic and perilymphatic compartments were perfused by Sulfo-NHS-LC-Biotin. Biotin was detected by FITC-conjugated streptavidin (green) in frozen sections of 6 week old mice. No sign of biotin entry into the stria vascularis compartment was found as shown by the arrow indicated that the tight junctions of marginal and basal cells are sealed in <i>Spns2^tm1a/tm1a</i> mice (<b><i>B</i></b>) compared with the control mice (<b><i>A</i></b>). <b><i>C,D</i></b>, Stria vascularis capillaries of young adult wildtype (<b><i>C</i></b>) and <i>Spns2^tm1a/tm1a</i> (<b><i>D</i></b>) mice following BSA-FITC injection into the tail vein, showing no evidence of leakage of the tracer (green) out of the capillaries. The increased branching of capillaries in these <i>Spns2^tm1a/tm1a</i> mice is also visible. Scale bars, 20 µm in <b><i>A–D</i></b>.</p

Progressive decrease in expression of Kcnj10, Kcnq1, Gjb2 and Gjb6 in <i>Spns2^tm1a/tm1a</i> mice.

<p><b><i>A–E</i></b>: At P14, Kcnj10 expression (green) of homozygotes was comparable to that of wildtype in apical turns, while in basal turns, some appeared normal (<b><i>B</i></b>) as wildtype (<b><i>A</i></b>), but some appeared largely reduced (<b><i>C</i></b>). At 5–6 weeks old, Kcnj10 labelling was absent in homozygotes (<b><i>E</i></b>). Acetylated α-tubulin (red) was used to label strial marginal cells in <b><i>D,E</i></b>. <b><i>F–I</i></b>: Whole mount preparations of the stria. Kcnq1 labelling (green) was detected at P14 in both homozygotes (<b><i>G</i></b>) and wild types (<b><i>F</i></b>), but it was absent from those marginal cells with enlarged cell boundaries in <i>Spns2^tm1a/tm1a</i> mice at 5–6 wks (<b><i>I</i></b>). Phalloidin (red) labelled filamentous actin to reveal the boundaries of marginal cells. <b><i>J–Q</i></b>: Gjb2 and Gjb6 were present in the fibrocytes of the spiral ligament in both wild type and <i>Spns2^tm1a/tm1a</i> mice at P14 (<b><i>J,K,N,O</i></b>). At 5–6 wks, expression was absent in the area behind the spiral prominence corresponding to the type II fibrocytes in homozygotes (<b><i>M</i></b> and <b><i>Q</i></b>) compared with wildtypes (<b><i>L</i></b> and <b><i>P</i></b>) of the same age. Root cells were labelled by acetylated α-tubulin (red) in <b><i>P,Q</i></b>. DAPI (blue) labelled the nuclei. Scale bar, 10 µm in <b><i>D,E</i></b>. 20 µm in <b><i>A–C</i></b>, <b><i>F–I,P,Q</i></b>. 50 µm in <b><i>J–O</i></b>.</p