Severe neurologic impairment in mice with targeted disruption of the electrogenic sodium bicarbonate cotransporter NBCe2 (Slc4a5 gene)

Abstract

The choroid plexus lining the four ventricles in the brain is where the majority of cerebrospinal fluid (CSF) is produced. The secretory function of the choroid plexus is mediated by specific transport systems that allow the directional flux of nutrients and ions into the CSF and the removal of toxins. Normal CSF dynamics and chemistry ensure that the environment for neural function is optimal. Here, we report that targeted disruption of the Slc4a5 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe2 results in significant remodeling of choroid plexus epithelial cells, including abnormal mitochondrial distribution, cytoskeletal protein expression, and ion transporter polarity. These changes are accompanied by very significant abnormalities in intracerebral ventricle volume, intracranial pressure, and CSF electrolyte levels. The Slc4a5(-/-) mice are significantly more resistant to induction of seizure behavior than wild-type controls. In the retina of Slc4a5(-/-) mice, loss of photoreceptors, ganglion cells, and retinal detachment results in visual impairment assessed by abnormal electroretinogram waveforms. Our findings are the first demonstration of the fundamental importance of NBCe2 in the biology of the nervous system.

FIGURE 1.

Insertional deletion of Slc4a5. A, restriction map of the retroviral gene trap vector, VICTR48. B, structural arrangement of 129 Sv/Ev^brd mouse gene Slc4a5; diagram of the mutated Slc4a5 allele after insertion. LTR, long terminal repeat. C, Western analysis. Lane 1, wild-type choroid plexus labeled with the NBCe2 antibody; lane 2, Slc4a5^−/− choroid plexus showing lack of labeling with the NBCe2 antibody; lane 3, HEK-293 cells expressing NBCe2 labeled with the NBCe2 antibody; lane 4, HEK-293 cells expressing NBCe2 labeled with the NBCe2 antibody preincubated with the immunizing peptide.

FIGURE 2.

A, immunolocalization of NBCe2 in wild-type CPE cells. B, CPE cells from Slc4a5^−/− mice fail to express NBCe2. C, Nomarski image corresponding to the immunofluorescence image shown in B. D and E, function of NBCe2 in wild-type CPE cells (n = 4) and lack of function in Slc4a5^−/− CPE cells (n = 6). A series of 400-ms voltage clamp pulses ranging from −80 to +40 mV with an increment of 20 mV was applied, and whole-cell current responses were recorded. Current-voltage (I-V) relationship of steady-state current in the absence and presence of HCO₃⁻ is presented. The results show that only wild-type CPE cells generate a positive current because of NBCe2-mediated transport in the presence of bicarbonate.

FIGURE 3.

Dissected fourth ventricle choroid plexus. A, wild-type mice; B, Slc4a5^−/− mice. The choroid plexus from Slc4a5^−/− mice have protrusions (arrows) that were not seen in control mice. Morphology of CPE cells (H&E stained cells) from wild-type mice (C) and Slc4a5^−/− mice (D) is shown. Cells from Slc4a5^−/− mice appear less columnar and have a granular cytoplasmic appearance. E, electron micrograph image of a CPE cell from wild-type mice showing apically localized mitochondria (arrow) in comparison with a cell from Slc4a5^−/− mice (F) where the mitochondria (arrows) are more evenly dispersed.

FIGURE 4.

Immunolocalization of H⁺/base transporters in CPE cells from wild-type and Slc4a5^−/− mice. AE2, wild-type (A); Slc4a5^−/− (B); NHE1, wild-type (C); Slc4a5^−/− (D); NBCn1, wild-type (E), Slc4a5^−/− (F); NCBE, wild-type (G), Slc4a5^−/− (H). Unlike wild-type CPE cells, which express NCBE basolaterally, in Slc4a5^−/− CPE cells, the transporter is localized basolaterally (1), apically (2), or bilaterally (3).

FIGURE 5.

Immunolocalization of Na⁺ pump subunits, NKCC1 and spectrin βII cytoskeleton protein. α1 Na⁺ pump subunit is expressed apically in wild-type CPE cells (A) and bilaterally (arrowhead) in CPE cells from Slc4a5^−/− mice (B). The β1 Na⁺ pump subunit was expressed apically in both wild-type (C) and Slc4a5^−/− CPE cells (D). Unlike wild-type CPE cells (E), CPE cells from Slc4a5^−/− mice fail to express the β2 Na⁺ pump subunit (F). NKCC1 that is expressed apically in wild-type CPE cells (G) is localized apically (arrow) and apically + intracellularly (arrowhead) in CPE cells from Slc4a5^−/− mice (H). Apical localization of spectrin βII in wild-type CPE cells (I) compared with its apical (arrow) and bilateral (arrowhead) expression in CPE cells from Slc4a5^−/− mice (J).

FIGURE 6.

MRI imaging (horizontal plane) of the lateral ventricles in wild-type (A) and Slc4a5^−/− mice (B) is shown. C, lateral ventricle volume assessed using MIPAV software and a three-dimensional analysis was performed using the MRIcro program. The ventricular volume in Slc4a5^−/− mice was significantly reduced; *, p < 0.05. D, intracranial pressure was also significantly reduced in Slc4a5^−/− mice; *, p < 0.05. Aquaporin 1 expression was unchanged in CPE cells from wild-type (E) versus Slc4a5^−/− mice (F). Similarly, aquaporin 4 expression was unchanged in wild-type (G) versus Slc4a5^−/− (H) ventricular ependymal cells.

FIGURE 7.

A–C, SD-OCT studies of wild-type (A) and Slc4a5^−/− (B and C) retinas. Slc4a5^−/− had retinal detachment of varying severity (B and C). D, ERG studies in wild-type and Slc4a5^−/− mice are as follows: a-wave amplitude (mean ± S.E.) as a function of stimulus intensity for wild-type mice (filled symbols) and Slc4a5^−/− mice (open symbols). These findings are consistent with the observations on EM of diminished photoreceptor number, shorter and dysmorphic outer segments, and inappropriate apposition of the photoreceptor outer segment tips with the RPE in Slc4a5^−/− mice; *, p < 0.05. E, ERG studies in wild-type and Slc4a5^−/− mice: a- to b-wave amplitude ratio demonstrating modest attenuation of b-wave amplitude for the Slc4a5^−/− relative to wild-type mice. Wild-type mice have higher ratios compared with Slc4a5^−/− mice showing that there is a small selective disruption of signal transmission from photoreceptor to bipolar retinal cells in Slc4a5^−/− mice; *, p < 0.05.

FIGURE 8.

A and B, immunolocalization of NBCe2 in the wild-type and Slc4a5^−/− retina (square box). A, localization of NBCe2 in the OPL. Inset, higher magnification of a region in the OPL of wild-type mouse illustrates horseshoe-shaped ribbon-like structures (arrowheads) in the OPL that are absent in Slc4a5^−/− mice (B). Faint nonspecific staining was detected in ganglion cells from both groups of mice. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; and GCL, ganglion cell layer. Scale bar, 10 μm. C–E, light microscopy of wild-type (C) and Slc4a5^−/− retina (D and E). In Slc4a5^−/− retina, a spectrum of abnormalities was detected. Some of the retina showed a translucent zone (D, arrows) between the apical region of the RPE and distal outer segments (ROS) that on electron microscopy is composed of densely pack RPE apical microvilli (G). The nuclei in the proximal region of the ONL are disorganized. A more severely affected retina is shown in E where a portion of the retina is detached (arrows) with photoreceptor cell death (reduced number of photoreceptor nuclei in the outer nuclear layer (ONL)). Large macrophages (arrowhead) are present among the damaged rod outer segments. F and G, electron microscopy of the retinal pigment epithelium/distal rod photoreceptor outer segment interface: wild-type (F) and Slc4a5^−/− mice (G). Apical microvilli from the RPE of wild-type mice (F) interdigitate in close association with distal rod outer segment tips. The apical surface of the RPE in Slc4a5^−/− mice (G) is displaced from distal rod outer segment tips by densely packed RPE apical microvilli (MV).

FIGURE 9.

Representative images of the optic nerve sections from wild-type (A) and Slc4a5^−/− mice (B). In Slc4a5^−/−mice, large irregular axons (arrows) and condensed axons with degenerated myelin sheaths (arrowheads) were observed. C and D, RGCs in whole-mount retinas were labeled with antibodies against Rbpms. Representative images showing reduced RGC density in Slc4a5^−/−mice (D) versus control (C). E, analysis of neuronal RGC density (averaged density) revealed a reduced number of RGCs in Slc4a5^−/− retinas. The superior temporal and inferior temporal quadrants at 1.5 and 2.0 mm from the optic nerve head were most affected (not shown). *, p < 0.05.