Endothelium-derived <i>Lrp6</i> is dispensable for retinal vascular development.

<p>Whole mount IF staining of ColIV (red) and FITC-Dextran perfusion showing (A) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. (B) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/fl</i> CKO mice. (C) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i> CKO mice. (D) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. All mice are 8 weeks of age. Scale bars = 100nm.</p

Conditionally restoring <i>Lrp5</i> in endothelial but not myeloid cells rescues retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A) FITC-Dextran perfusion showing distorted NFL and IPL vessels and lack of OPL vascular development in <i>Lrp5</i>^{<i>a214v(n)/-</i>} hypomorph mice (lower panels) compared to controls (upper panels) both in retinal whole mount (4w) and cross section (2m) images. (B) <i>VE-Cad-Cre;Lrp5</i>^{<i>a214v(n)/+</i>} (upper left panels, FITC-Dextran perfusion), <i>VE-Cad-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (lower left panels, FITC-Dextran perfusion) and <i>Flk1-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (upper right panels, red: ColIV, green: IB4) mice all developed a normalized three-tier retinal vascular structure, while <i>LysM-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (lower right panels, red: ColIV) mice displayed similar retinal vascular abnormalities compared to control <i>Lrp5</i>^{<i>a214v(n)/-</i>} mice (A, lower panels). Quantification of vascular branch points in OPL is shown in graph at right; **P<0.01, ns not significant. All mice are between 4 to 5 weeks of age except otherwise labeled. Scale bars = 100nm.</p

Loss of <i>Lrp5</i> causes retinal hypovascularization and neovascularization.

<p>(A) ColIV whole mount IF staining showing retinal vessels of <i>Lrp5</i>^<i>-/-</i> and control mice at P9 and P30. Quantification of vascular sprout numbers at P5 shown at right. (B) Adult <i>Lrp5</i>^<i>-/-</i> retinas showing persistent hyaloid vessels (black arrows, CD31 IHC staining), aneurysms (open arrow, CD31 IHC staining), neovascular overgrowth (white arrows, fibronectin IF staining) and lack of IPL and OPL vascular development (lower panels, green: FITC-Dextran perfusion). (C) FITC-Dextran perfusion (green) showing retinal vascular leakage (white arrows) in adult <i>Lrp5</i>^<i>-/-</i> mice compared to control. Scale bars = 100μm. (D) EM analysis of endothelium of <i>Lrp5</i>^<i>-/-</i> and control retinas at P5, P8 and P30. Arrows point to area of endothelial fenestration. Scale bars = 500nm. (E) Total amounts of VEGF protein in retinas of <i>Lrp5</i>^<i>-/-</i> and control mice at P5, P8 and P30. Each ELISA was done in duplicate and normalized to total retinal protein amount. <i>n</i> = 9, 5, 12 for controls (at P5, P8, P30) and 9, 8, 8 for <i>Lrp5</i>^<i>-/-</i> (at P5, P8, P30). * <i>p</i><0.05, ** <i>p</i><0.01. Data are represented as means ± SD. Ctrl, control.</p

Retinal vascular phenotype in mice with conditional knockout of <i>Lrp5</i> in myeloid/microglial cells.

<p>(A) Whole mount IF staining with IB4 (blue) or ColIV antibody (right panel, green) showing distribution of <i>LysM-Cre;tdTomato</i>⁺ (red) myeloid and microglial cells in retinas at P5, P8 and 6w. Most <i>LysM-Cre;tdTomato</i>⁺ myeloid cells were also F4/80⁺ (green) (white rectangle in left panel indicates area shown at higher magnification in the upper right corner inset). (B) Localization of <i>LysM-Cre;tdTomato</i>⁺ myeloid cells in three adult (6w) retinal vascular beds. Green: ColIV. (C) Retinal vasculature of <i>LysM-Cre;Lrp5</i>^<i>fl/-</i> CKO mice (12w). Red: IB4. (D) Distribution of <i>CD11b-Cre;tdTomato</i>⁺ (red) myeloid and microglial cells in retinas at P6 (left and mid panels, green: IB4) and 4w (right panel, cross section). Blue: Hoechst. (E) Localization of <i>CD11b-Cre;tdTomato</i>⁺ signals (red) in myeloid, microglial, Müller glial and perivascular macrophage cells in adult (4w) retina. Blue: IB4. White rectangle in right panel outlines cell shown at higher magnification in upper right corner inset. (F) Retinal vasculature of <i>CD11b-Cre;Lrp5</i>^<i>fl/-</i> CKO mice (4w). Green: FITC-Dextran perfusion. Scale bars = 100nm.</p

Conditional knockout of <i>Lrp5</i> with <i>Tie2-Cre</i> but not <i>VE-Cad-Cre</i> recapitulates retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A and B) ColIV IF staining (red) and FITC-Dextran perfusion (green) showing adult retinal vasculature in <i>VE-Cad-Cre;Lrp5</i>^<i>fl/-</i> and <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i> CKO mice (4w) compared to <i>Lrp5</i>^<i>fl/fl</i> control. Arrows: hyaloid vessels; open arrows: neovascular tufts; arrowheads: neovascular overgrowth. At right: Quantification of vascular branch points in OPL at 4w; ns not significant. (C) TdTomato signals in <i>VE-Cad-Cre;tdTomato</i> (8w) and <i>Tie2-Cre;tdTomato</i> (7w) mice showing predominant endothelial expression of <i>VE-Cad-Cre and Tie2-Cre</i> in the NFL, IPL and OPL of the retina. Note that <i>VE-Cad-Cre</i> is also widely expressed in myeloid cells around the vessels in the NFL (upper left panel, also see D). Arrows point to IB4 (green) positive vascular area with negative <i>VE-Cad-Cre;tdTomato</i> signals in a P5 mouse indicating incomplete <i>VE-Cad-Cre</i> recombination. (D) Myeloid and microglial localization of <i>VE-Cad-Cre;tdTomato</i> and <i>Tie2-Cre;tdTomato</i> signals in P5 (left and mid-left panels) and adult (right and mid-right panels) retinas. Arrows point to myeloid cells and arrowheads point to microglial cell. Green: F4/80 IF staining. Scale bars = 100nm.</p

<i>Flk1-Cre</i>^{<i>Breier</i>} is specifically expressed in endothelial cells and conditional knockout of <i>Lrp5</i> with <i>Flk1-Cre</i>^{<i>Breier</i>} recapitulates retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A) Specific endothelial location of <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;tdTomato</i> signals in developing retinas (P5, upper panels) overlapping with IB4 IF signals (blue). F4/80 IF staining (green) showing that most macrophages were tdTomato negative. In adult retina, <i>Flk1-Cre</i>^{<i>Breier</i>} was specifically expressed in all three layers of the retinal vascular beds (lower panels) with no signs of any myeloid cell expression. Penetrance of <i>Flk1-Cre</i>^{<i>Breier</i>} expression in ECs could also be incomplete as shown in (D). (B) Specific endothelial location of <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;tdTomato</i> signals in adult (4w) bone marrow, cortical bone and skeletal muscle. M: muscle; CB: cortical bone; BM: bone marrow. Blue: Hoechst. (C) Whole mount IF staining of ColIV (red) and IB4 (green) showing retinal vasculature in adult (4w) <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;Lrp5</i>^<i>fl/-</i> CKO mice with disorganized NFL vessels, vertical vessel branches terminating in ball-like structures in the IPL and lack of OPL vascular bed. Arrow points to persistent hyaloid vessels. Note that regions with vascular abnormalities often had patchy normal-looking areas located in the neighborhood. In the selected OPL image, an area with well-developed vessels is next to another with no vascular development. (D) Whole mount IF staining of IB4 (green) showing that normally developed vascular areas in <i>Flk1-Cre;Lrp5</i>^<i>fl/-</i><i>;tdTomato</i> retinas (8w) included many <i>Flk1-Cre;</i>tdTomato negative vessels (arrows), whereas abnormal vessel structures were all tdTomato positive (open arrows). Scale bars = 100μm.</p

Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

<div><p>More than 90% of the cases of Parkinson’s disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using <i>Xenopus</i> oocytes injected with <i>Aqp9</i> cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP⁺). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite—another parkinsonogenic toxin. Conversely, targeted deletion of <i>Aqp9</i> in mice protects nigral dopaminergic neurons against MPP⁺ toxicity. A protective effect of <i>Aqp9</i> deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP⁺ <i>in vitro</i> and in mice subjected to intrastriatal injections of MPP⁺ <i>in vivo</i>. Seven days after intrastriatal MPP⁺ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in <i>Aqp9</i> knockout mice compared with 67% in WT littermates. Our results show that AQP9 –selectively expressed in catecholaminergic neurons—is permeable to MPP⁺ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysiology of Parkinson’s disease.</p></div

<i>Xenopus</i> oocytes expressing AQP9 reveal higher uptake of MPP⁺.

<p><i>Xenopus</i> oocytes expressing AQP4 or AQP9 and uninjected oocytes were exposed to ¹⁴[C]urea or ³[H]MPP⁺. Data were obtained as counts per minute (CPM)/oocyte and averaged for each construct. The uptake was normalized to that of the AQP4-expressing oocytes prior to averaging across the n = 7 experiments with five to ten oocytes included for each experimental condition. Compared with uninjected oocytes and AQP4 expressing oocytes, oocytes expressing AQP9 accumulate significantly higher amounts of urea as well as MPP⁺. Bars are mean ± SEM; *p<0.05, ***; p<0.001.</p

Semiquantitative PCR analyses of gene expression in <i>Aqp9</i>^-/- and WT mice brain.

<p>A) Semi-quantitative Real-Time PCR revealed significantly higher <i>Aqp9</i> mRNA levels in the midbrain and striatum than in the neocortex. The level of <i>Aqp9</i> mRNA in neocortex is 59% of that in midbrain (p = 0.002). The level of <i>Aqp9</i> mRNA in the <i>Aqp9</i>^-/- mice was close to the detection limit (n = 7 for each genotype). B) Representative DNA agarose gel electrophoresis of midbrain samples from WT and <i>Aqp9</i>^-/- mice (upper panel), and of three different regions in WT mice (lower panel). These data support the PCR analysis in A. C) In order to rule out that the reduced dopaminergic cell loss in the <i>Aqp9</i>^-/- mice could be attributed to compensatory up- or downregulation of other genes, an analysis was done of the expression levels of <i>Aqp4</i>, <i>Gfap</i>, <i>Kir4</i>.<i>1</i>, <i>mTOR</i>, <i>Prph</i>, <i>Cat</i>, <i>Ppard</i>, <i>Slc6a3 (DAT)</i>, <i>Drd2</i>, <i>Bcl2</i>, <i>Bax</i> and <i>Sod2</i>. The relative levels of these transcripts did not differ between <i>Aqp9</i>^-/- (n = 7) and WT animals (n = 7). D-H). For selected genes the expression levels were analyzed in the treated and untreated hemispheres. In both groups of animal, the transcript levels of <i>Drd2</i> were lower in the striatum on the injected side than in the striatum on the contralateral side (E). In contrast, in both groups of animals, the level of <i>Bax</i> was higher in the ipsilateral striatum than in the contralateral one (H). The values indicated in the graphs for <i>DAT</i>, <i>Bcl2 and Sod2</i> show the values for midbrain, and the values for <i>Drd2</i> are for striatum. All values are relative to the values for the corresponding samples from the control hemisphere of the saline treated mice. Bars are mean ± 2 SEM; **p<0.005.</p

HEK293 cells expressing EGFP-<i>h</i>AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP-<i>h</i>DAT.

<p>A-C) Native HEK293 cells and HEK293 cells expressing EGFP-<i>h</i>AQP9 or YFP-<i>h</i>DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP-<i>h</i>AQP9 and YFP-<i>h</i>DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP-<i>h</i>AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP-<i>h</i>AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP-<i>h</i>AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP-<i>h</i>DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p<0.05, **p<0.01, ***p<0.001; crosses: significantly different from previous data point: ++ p<0.01.</p