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First published online 25 November 2008
doi: 10.1242/jcs.035758

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AQP4 knockout impairs proliferation, migration and neuronal differentiation of adult neural stem cells

Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China

View larger version (58K): [in this window] [in a new window]   Fig. 1. AQP4 is required for ANSC self-renewal and proliferation in vitro. (A) Morphological examination showed the reduced number and size of Aqp4–/– primary, secondary and tertiary neurospheres compared with the Aqp4+/+ control. Primary neurospheres were initiated from dissociated adult rostral periventricular tissue. Cells obtained by dissociating primary neurospheres proliferated and yielded secondary neurospheres. Single cells obtained after dissociation of secondary neurospheres proliferated and yielded tertiary neurospheres. Scale bar: 200 µm. (B,C) Neurosphere assay. The number and size of neurospheres were determined after 7 days for primary, secondary and tertiary neurospheres (*P<0.05 vs Aqp4+/+, n=3). (D,E) BrdU incorporation assay. The percentage of BrdU+ cells in Aqp4–/– ANSCs was significantly lower than that in Aqp4+/+ cells (*P<0.05, n=4).

View larger version (28K): [in this window] [in a new window]   Fig. 2. AQP4 modulates the cell cycle characteristics of cultured SVZ ANSCs. (A) Representative flow-cytometric histograms of DNA content of Aqp4+/+ and Aqp4–/– ANSCs population. (B) There was no significant difference in the proportion of cells at G0-G1 phase and S phase between Aqp4+/+ and Aqp4–/– ANSC populations. The proportion of G2-M phase cells was increased in Aqp4–/– ANSCs compared with the wild type (*P<0.05, n=3). Moreover, AQP4 knockout significantly increased the basal apoptosis of ANSCs (*P<0.05, n=3).

View larger version (69K): [in this window] [in a new window]   Fig. 3. In vitro neurosphere migration assay. (A) SVZ neurospheres cultured on poly-L-ornithine and laminin-coated coverslips migrate outwards radially. Scale bar: 200 µm. (B) 48 hours after adhesion, AQP4 knockout significantly decreased the distance of radial migration of SVZ neurospheres compared with the Aqp4+/+ control (*P<0.05).

View larger version (81K): [in this window] [in a new window]   Fig. 4. AQP4 knockout impairs neuronal differentiation of ANSCs. (A) ANSCs derived from SVZ could generate into neurons (TUJ1), astrocytes (GFAP) and oligodendrocytes (GalC) after 7 days of culture in differentiation medium. Scale bar: 100 µm. (B,C) The proportion of TUJ1+ cells in the Aqp4–/– cell population was significantly lower than that of Aqp4+/+ cells (*P<0.05, n=3). Scale bar: 50 µm. (D) No significant difference was found in the proportion of GFAP+ cells between Aqp4–/– and Aqp4+/+ cell populations.

View larger version (82K): [in this window] [in a new window]   Fig. 5. AQP4 knockout alters spontaneous Ca2+ oscillations and suppresses high concentration KCl-induced Ca2+ transient in ANSCs. (A) Double immunocytochemistry of nestin (green) and AQP4 (red) in cells migrated out of adherent neurospheres 24 hours after plating. (B) Spontaneous Ca2+ oscillations recorded from three Aqp4+/+ and Aqp4–/– ANSCs (the oscillations of each cell are represented by a different color) loaded with fluo-3 AM. (C) Statistical analysis of data obtained from three independent experiments indicated that the mean amplitude (F/F0) of spontaneous Ca2+ oscillations in Aqp4–/– ANSCs was significantly lower than that of Aqp4+/+ control (*P<0.01). The frequency of spontaneous Ca2+ oscillations recorded from Aqp4–/– ANSCs was significantly enhanced compared with that of Aqp4+/+ control (*P<0.01). (D) Sequence of images recorded over 90 seconds from Aqp4+/+ and Aqp4–/– ANSCs loaded with fluo-3 AM following KCl-induced depolarization. (E) Fluo-3 fluorescence expressed as F/F0; increased fluorescence indicates elevated [Ca2+]i. There was a significant inhibition of peak amplitudes and no significant sustained phase durations in Aqp4–/– ANSCs compared with Aqp4+/+ control. (F) Peak amplitudes of the KCl-induced Ca2+ transient show a significant decrease in Aqp4–/– ANSCs compared with that in the Aqp4+/+ control (*P<0.001). Scale bars: 50 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. AQP4 knockout downregulates the expression of Cav1.2 and connexin43 in ANSCs. (A) RT-PCR analysis shows Cav1.2, Cav3.1 and TRPC1 mRNA is present in the ANSCs. The transcripts of Cav1.3, Cav3.2 and Cav3.3 were not detectable. (B) In Aqp4–/– ANSCs, the mRNAs of genes encoding Cav1.2 and Cav3.1 were significantly decreased when normalized to GAPDH (*P<0.05, n=3). There was no significant inhibition in the transcription of TRPC1 in Aqp4–/– ANSCs compared with the Aqp4+/+ control. (C) Western blot confirms that AQP4 knockout decreased the expression of Cav1.2 channel protein in the ANSCs compared with the wild type (*P<0.05, n=3). Cav3.1 channel protein was not detected. (D) Western blotting (top panel) showing that the expression level of Cx43 protein in Aqp4–/– ANSCs decreased to 39% of the level in the Aqp4+/+ genotype (lower panel) (*P<0.01, n=3).

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