9 research outputs found
Recommended from our members
A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1
A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.00
Recommended from our members
Impaired AQP2 trafficking in Fxyd1 knockout mice: A role for FXYD1 in regulated vesicular transport
The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes
Washout of dDAVP revealed rapid redistribution of AQP2 in slices of inner medulla from <i>Fxyd1</i> knockout mice.
<p>Slices from WT (a) and KO mice (b) treated with dDAVP for 15 min as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188006#pone.0188006.g005" target="_blank">Fig 5</a> were washed in HBSS for 60 min. Slices were fixed with PLP, sectioned, and stained for AQP2. The absence of FXYD1 resulted in less juxta-apical and more heterogeneous localization of AQP2 in kidney from KO mice (b). Arrows point to regions with AQP2 in proximity to the basolateral membrane. The images were enhanced and sharpened by subjecting the entire field to the high-pass filter in Adobe Photoshop. Bar, 10 μm.</p
AQP2 acute response to vasopressin differed in slices of inner medulla from wild type and <i>Fxyd1</i> knockout mice.
<p>Kidney slices from WT (a-c) and KO (d-f) male mice were fixed without treatment (a,d), incubated in HBSS to wash out endogenous VP (b,e), and then treated with dDAVP for 15 min (c,f). Slices were post-fixed with 2% PLP and sectioned. The distribution of AQP2 was monitored by immunofluorescence. The absence of FXYD1 reduced shuttling of AQP2 between intracellular vesicles and subapical spaces and apical membrane. This figure is representative of 3 independent experiments. Nuclei were labeled with Dapi. Bars, 10 μm.</p
Flotation experiments revealed a difference in distribution of AQP2 in detergent-resistant membranes (DRM) from WT and knockout mouse.
<p>Membranes from inner medulla from wild type (solid lines in A and B) or FXYD1 KO male mice (dashed lines in A and B) treated with either vehicle (A) or dDAVP (B) for 1 hour were solubilized with 1% Triton, 30 min, 4°C, and separated on a step sucrose gradient of 5, 30, and 40% sucrose. DRM float near the top of the gradient (peaks 1 and 2). The distribution of AQP2 was quantified on Western blots with a cooled-CCD imager and expressed as % of total recovered AQP2 in each fraction. The data are mean ± SEM of four experiments. (A) Under basal conditions, the DRM AQP2 fraction from vehicle-treated knockout mice peaked in peak 1, while WT DRM appeared in peak 2. Roughly half of the AQP2 was in denser membranes (peak 3). (B) Stimulation with dDAVP caused redistribution of DRM AQP2 from the KO to peak 2. (C) Vasopressin-stimulated recruitment of AQP2 from peak 3 to DRM membranes in peak 2 occurred in WT but not in KO. (**) The difference is significant by Student’s <i>t</i>-test, P < 0.005, n = 4.</p
FXYD1 is implicated in urinary concentration.
<p>Box and whisker plot of the osmolality of daytime (afternoon) samples of male mouse WT and knockout (KO) urine at baseline and after water deprivation, showing a difference in baseline osmolality but the near-normal ability of the KO to concentrate its urine. The asterisks indicate a P value of <0.0001, 2-tailed Student’s <i>t</i>-test. When calculated as average ± SEM the results were as follows. WT control conditions, 2,019 ± 143, n = 20; KO control conditions 1,161 ± 123, n = 21; Student’s <i>t</i>-test, P < 0.0001. WT after 36 hours water deprivation, 4,224 ± 140, n = 8; KO water deprivation, 3,716 ± 383, n = 8; <i>t</i>-test, P = 0.26. Female mice were also tested in control conditions, and the results for baseline osmolality were WT, 2,169+/-92, n = 7; KO, 1,085+/-108, n = 7, <i>t</i>-test P < 0.001.</p
AQP2 abundance was reduced in FXYD1 knockout mice.
<p>(A, B) <i>Fxyd1</i><sup>-/-</sup> mice had a lower abundance of AQP2 in inner medulla. Inner medulla from WT and KO mice was obtained as lysates and tested on blots with specific antibodies [representative blot in (A)]. Both core (c) and glycosylated (g) species of AQP2 were reduced in KO animals. (B) quantification of results of three experiments. (C, D) Blot quantification of the relative changes in inner medullary AQP2 levels after 2 hour treatment of mice with dDAVP, 3 experiments. Stimulation with dDAVP resulted in a similar increase of AQP2 recovered in pelleted crude membranes in both WT (C) and KO (D). Bars are ± S.E.M. and significance was evaluated by Student’s <i>t</i>-test.</p
The daytime defect in urine concentration in Fxyd1<sup>-/-</sup> mice was compensated over 24 h.
<p>The daytime defect in urine concentration in Fxyd1<sup>-/-</sup> mice was compensated over 24 h.</p
Vasopressin induced trafficking of FXYD1 in wild type IMCD <i>in vivo</i>.
<p>Wild type mice were injected with dDAVP (1 μg/kg) and sacrificed at 0 time (a-c), 4 hours (d-f), and 16 hours (g-i) post-injection. Fixed kidneys were sectioned, and slides were stained with antibodies against AQP2 (a,d,g) and PLM-C1 antibodies against FXYD1 (b,e,h). dDAVP stimulated the trafficking of FXYD1 from an intracellular location towards apical membrane. Co-localization of FXYD1 and AQP2 at apical membrane is seen in yellow after 4 hours (f). This figure is representative of 3 independent experiments. Nuclei were labeled with TO-PRO-3. Bars, 10 μm.</p