Water Channels Aquaporin 4 and -1 Expression in Subependymoma Depends on the Localization of the Tumors

<div><p>Background</p><p>We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior.</p><p>Methods and Results</p><p>Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels.</p><p>Conclusions</p><p>Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1.</p></div

Freeze fracture electron micrograph.

<p>A Replica from tissue of patient 5: SE (infratentorial), no OAPs were found, B: Healthy astrocytic endfoot membrane of rat cortex is covered with OAPs. Bar 250 nm.</p

Staining for MMPs in SE tissues.

<p>Infratentorial (A, C, E, patient 5) and supratentorial (B, D, F, patient 9) SE tissues were stained with antibodies against MMP2 (A, B), MMP3 (C, D), and MMP9 (E, F). The inserts with double letters show a higher magnification as indicated. None of the immunostains showed significant immunoreactivity. For controls, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131367#pone.0131367.s003" target="_blank">S3 Fig</a>.</p

Primer sequences and product sizes.

<p>*The AQP4 Primers were designed to amplify both isoforms of AQP4 (M1+M23)</p><p>Primer sequences and product sizes.</p

Patient history.

<p>Patient history.</p

Primary antibodies used in human subependymomas.

<p>Primary antibodies used in human subependymomas.</p

Immunhistological staining of agrin (A-D) and alpha-dystroglycan (E-H) in SE.

<p>Neither infratentorial SE (A, C, E, G, patient 5) nor supratentorial SE (B, D, F, G, patient 9) were positive for agrin or dystroglycan, normally found around the blood vessels (for positive control see supplement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131367#pone.0131367.s003" target="_blank">S3 Fig</a> Bar 1000 and 50μm.</p

Immunofluorescent double staining for aquaporins in SEs.

<p>Double staining of GFAP- (green) and AQP4 (red) in infratentorial (A, patient 5) and supratentorial (B patient 6) SE. Dapi stained nuclei in blue. Double staining of AQP4 (red) and AQP1 (green) in infratentorial (C patient 4) and supratentorial (D patient 6) SE. AQP 1 and AQP4 positive cells are more or less evenly distributed in infratentorial tumors, in contrast they were found only in the border region of supratentorial tissue. Bar: 50μm (A, B, C); 100μm (D).</p

PCR analysis of AQP4 and AQP1 infratentorial (patients 1–5 or lane 1–5 respectively) and supratentorial (patients 6–10 or lane 6–10) respectively SE.

<p>A: In every location AQP4 (Exon 4–5) was expressed. Lane 11: negative control (H₂O), lane 12 positive control (lung). HPRT lane 1–12. B: In every location AQP1 was expressed. The infratentorial SE samples showed very distinct bands in the gel, whereas the AQP1 expression in supratentorial SEs varied and where less distinct. Lane 11: negative control (H₂O), lane 12 and 13 positive control (Normal Brain). HPRT1 lane 1–13.</p

Highly schematic diagram indicating the location of subependymomys (SEs) in the human brain.

<p>The main findings on AQP distributions are also indicated.</p