CLCN7 and the chorid plexus

This paper is actually a great introduction to the CIC7, the gene product of the CLCN7 gene. The choroid plexus is a network of capillaries and specialized epithelial cells called ependymal cells that regulate the production and composition of cerebrospinal fluid (CSF)

  1. CIC isoforms, including CIC7may help maintain the pH of the CSF
  2. The Na+/H+ exchanger, NHE6 sodium hydrogen exchanger may help in this task.

Could impaired regulation of CSF pH be part of the pahtology of NLCN7 mutation pathologies?

Damkier HH, Christensen HL, Christensen IB, Wu Q, Fenton RA, Praetorius J. The murine choroid plexus epithelium expresses the 2Cl/H+ exchanger ClC-7 and Na+/H+ exchanger NHE6 in the luminal membrane domain. Am J Physiol Cell Physiol. 2018 Apr 1;314(4):C439-C448.

This paper is rather unusual in that much can be understand just by looking at the figures and reading the legends.

Figure 1. FACS isolation of mouse choroid plexus epitehlial CPE cells.

The choroid plexus was surface‐labeled with fluorescent concanavalin A and enzymatically digested into cell suspensions.  To learn more about fluorescent activated cell sorting, consult this site.

  • (A) The left panels show transmitted light micrographs of a cell suspension at low and high magnification, while the right panel shows a high magnification fluorescence micrograph of the same cells.
  • (B) The left panel shows the side and forward scatter (SSC and FSC, respectively) data from FACS isolated CPE cells. The frame represents the gating for further cell analysis. The middle panel shows selection of green fluorescence (FITC) after detection of singlets, while excluding damaged cells based on propidium iodide uptake (PI), as indicated by the boxes. The right panel exemplifies a recount of the isolated FITC positive cell suspension.  PI is a dye used to detect the nuclei of dead cells.

2. Expression of NHE6 in the CPE.

  • A) RT-PCR analysis of NHE6 and NHE1 mRNA expression in FACS isolated CPE cells. RT-PCR products of the expected sizes were observed in large FITC positive cells (Large FITC+), small FITC positive cells (Small FITC+), undigested choroid plexus (Ch.Plex.), kidney cortex, and cerebral cortex (Cerebr.). Only NHE1 mRNA was detected in FITC negative cells (FITC- cells).
  • B) RT-PCR analysis of the expression of other NHE mRNAs in the CP. The analysis did not reveal expression of any other NHE forms (NHE2, NHE3, NHE4, NHE5, NHE7, NHE8, or NHE9) in the undigested CP (lane 3), while control tissues were positive for the relevant NHE isoform.
  • C) Immunoblotting of choroid plexus (CP) and kidney control (Kid) samples for NHE6 protein. Immuno-reactive bands of the expected size of approximately 70 kDa and an additional 165 kDa band were observed in both samples (arrowheads).
  • D) Immunohistochemical staining of cryostat sections of the CP for NHE6 protein (green). The right panel is a higher magnification micrograph of the tissue shown in the left panel. Immunoreactivity was most pronounced in the luminal membrane domain.

E) Double immunofluorescence labeling of cryostat sections for NHE6 (green) and the Na+,K+-ATPase (red) at two magnifications. Nuclei are stained blue. VL: IVth ventricle lumen, LS: luminal surface, BL: basolateral side. The right panel is a differential interference contrast micrograph of the cells shown in the middle panel.

Figure 3. Expression of Cl/H+ exchangers in the CPE.

There are a bunch of them leading us to question whether a mutation in CLCN7 is really that important when there may be backups.

  • RT-PCR analysis of ClC-3, ClC-4, ClC-5 and ClC-7 mRNA expression in FACS (Concavalin A-Fitc  cell sorting) isolated CPE cells. PCR products of the expected sizes were observed for all four ClC proteins in FITC positive cells (FITC+), undigested choroid plexus (Ch.Plex.), and kidney control samples.
  • B) Immunohistochemical staining of paraffin sections of the CP for ClC-7 protein (green). The right panel is a higher magnification micrograph of similar tissue as the one shown in the left panel. Immunoreactivity was restricted to the luminal membrane domain and its brush border.
  • C) Double immunofluorescence labeling of similar sections for early endosome antigen EEA1 (Green) and late endosome/lysosome marker cathepsin D (Red) overlaid onto a differential interference contrast image. The staining patterns for the endo-lysosomal markers are quite punctate and distributed in the entire cytoplasm compared to the NHE6 labeling in the previous panel.  Nuclei are stained blue and the fluorescence signals were overlaid onto a differential interference contrast image. VL: IVth ventricle lumen, LS: luminal surface, BL: basolateral side.

Figure 4. Evidence for Cl/H+ exchange in isolated choroid plexus cells.

What makes this figure so interesting is that it brings home the point that CIC7 is bi-directional in which way it transports H+ and Cl-.

  • A) The CP was enzymatically digested into small clusters of epithelial cells and loaded with the pH sensitive dye BCECF. Recordings of pHi were performed without CO2/HCO3-. The trace shows a calibrated single cell recording of pHi when HEPES-buffered bath solution (HBS) was shifted from Cl–free (0Cl-HBS) to Cl-containing HBS and back to Cl-free solution, as indicated.
  • B) Summarized data of the pHi increase caused by addition of Cl- in the bath solution and the pHi decrease resulting from Cl removal (n=4).
  • C) Model for the net movement of H+ and Cl across a cell membrane in the two experimental settings: addition of Cl (top half) and removal of Cl (bottom half) from the bath. The expected pH changes and changes in membrane potential by the respective maneuvers are indicated.
  • D) Single cell trace showing the relative change in DiBAC fluorescence upon manipulations of the Cl- concentration and of exposure to 50 mM K+. The inset is an example micrograph of the cellular DiBAC signal.  DiBAC reacts to depolarization by increases in fluorescence intensity and by decreases in fluorescence when cells hyperpolarize.
  • E) Summarized data of the changes in DiBAC fluorescence caused by removal of Cl- (transient decrease and sustained increase, respectively), by addition of Cl- (HBS), and by 50 mM K+ (Hi-K HBS) (n=6).
  • F) Similar data obtained in the presence of the CO2/HCO3- buffering system (transient decrease and sustained increase, respectively) upon removal of Cl- (n=5). * indicates statistical significance.

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