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

HEK293 cells expressing <i>h</i>AQP9 or <i>h</i>DAT reveal higher sensitivity to MPP⁺.

<p>A-B) Immunofluorescence images of HEK293 cells expressing EGFP-<i>h</i>AQP9 (A) or YFP-<i>h</i>DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) 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 MPP⁺ (four 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. Native HEK293 cells show sensitivity to MPP⁺ only at very high concentrations (~100 μM). Cells expressing <i>h</i>DAT become sensitive at 1 μM MPP⁺, compared with 0.1 μM for cells expressing <i>h</i>AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP-<i>h</i>DAT expressing (F) and EGFP-<i>h</i>AQP9 expressing HEK293 cells (G) are shown. 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: ++<0.01.</p

HPLC analysis of dopamine and its metabolites.

<p>A-C) After unilateral injections of MPP⁺ in the striatum, the ipsilateral striatum shows a significant reduction in the concentration of DA (A), HVA (B) and DOPAC (C) compared to the contralateral hemisphere (<i>Aqp9</i>^-/-, n = 6; WT, n = 6). The ipsilateral reduction in HVA is significantly more pronounced in WT mice than in <i>Aqp9</i>^-/- mice (p = 0.038). Corresponding p-values for DA and DOPAC are 0.063 and 0.059, respectively. Bars are mean ± 2 SEM; *p<0.05, ** p<0.01, ***p<0.001.</p

Deletion of <i>Aqp9</i> protects against MPP⁺ toxicity <i>in vitro</i>.

<p>A) Photomicrograph (left) and schematic representation (right) of a representative midbrain slice. Site of MPP⁺ application is indicated in red. B-E) Immunofluorescence staining of representative midbrain slices from WT (B, C) and <i>Aqp9</i>^-/- mice (D, E) showing TH-positive neurons in the MPP⁺ treated side (ipsilateral; B, D), and in the control side (contralateral, C, E). Note the extensive loss of ipsilateral TH-positive neurons in the WT slice (B). F-H) Quantitative analyses of the TH-positive cell count in slices treated with 30 μM MPP⁺ (F, G) show significant loss of TH-positive neurons in the ipsilateral hemisphere of the WT slice (n = 8) (F). No significant difference was observed between the ipsi- and contralateral hemisphere of <i>Aqp9</i>^-/- slices treated with 30 μM MPP⁺ (n = 7) (G), WT slices treated with a combination of 60 μM MPP⁺ and 100 μM phloretine (n = 11) (H), or WT slices treated with saline (n = 8) (G). In WT mice, the TH-positive cell count contralateral to MPP⁺ application (F) was lower than the TH-positive cell count in saline treated slices (H), suggesting spillover of MPP⁺ from the ipsilateral side. RRF: retrorubral field; Aq: aqueduct; V: ventricle; A9: population of dopaminergic neurons. Bars are mean ± SEM; ***p<0.001. Scale bar: 100 μm; scale bar inset: 50 μm.</p

Deletion of <i>Aqp9</i> protects against MPP⁺ toxicity <i>in vivo</i>.

<p>A) Representative midbrain sections of WT and <i>Aqp9</i>^-/- mice, seven days after treatment with unilateral striatal injection of 7.5 μg MPP⁺. Cell bodies and processes of the dopaminergic neurons are identified by TH immunostaining. The visible reduction of the TH immunostained cell bodies in the ipsilateral SNpc is more pronounced in WT than in <i>Aqp9</i>^-/- mice. B) Quantitation of the TH-positive neurons in SNpc is described in the text. TH-positive cell loss, calculated as [(n contra—n ipsi): (n contra)] is significantly lower in <i>Aqp9</i>^-/- mice, where 47.59% of the cells are lost, compared to 67.02% in WT littermates (p<0.001). C) Compared with WT mice, <i>Aqp9</i>^-/- mice show a significantly higher count of TH-positive neurons on the injected side (p<0.001). D) Animals treated with saline showed no significant loss of TH-positive cells in SNpc, regardless of genotype. E) In WT mice, VTA showed a slight but statistically significant decrease in number of TH-positive neurons on the ipsilateral side (p = 0.033). No change was observed in VTA of <i>Aqp9</i>^-/- mice. The MPP⁺ treated group consisted of 15 animals (<i>Aqp9</i>^-/-, n = 8; WT, n = 7), and the saline group consisted of six animals (<i>Aqp9</i>^-/-, n = 3; WT, n = 3). ML, medial lemniscus. Bars are mean ± 2 SEM; n = 6; *p<0.05, **p<0.01, ***p<0.001. Scale bar, 1000 μm; scale bar inset, 20 μm.</p