A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus

Abstract

Idiopathic normal pressure hydrocephalus (iNPH) is an enigmatic neurological disorder that develops after age 60 and is characterized by gait difficulty, dementia, and incontinence. Recently, we reported that heterozygous CWH43 deletions may cause iNPH. Here, we identify mutations affecting nine additional genes (AK9, RXFP2, PRKD1, HAVCR1, OTOG, MYO7A, NOTCH1, SPG11, and MYH13) that are statistically enriched among iNPH patients. The encoded proteins are all highly expressed in choroid plexus and ependymal cells, and most have been associated with cilia. Damaging mutations in AK9, which encodes an adenylate kinase, were detected in 9.6% of iNPH patients. Mice homozygous for an iNPH-associated AK9 mutation displayed normal cilia structure and number, but decreased cilia motility and beat frequency, communicating hydrocephalus, and balance impairment. AK9+/- mice displayed normal brain development and behavior until early adulthood, but subsequently developed communicating hydrocephalus. Together, our findings suggest that heterozygous mutations that impair ventricular epithelial function may contribute to iNPH.

Keywords: AK9; motile cilia; normal pressure hydrocephalus; whole exome sequencing.

Fig. 1.

iNPH-associated mRNA expression in the ventricular region of the mouse brain. In situ hybridization showing expression of mRNAs for eight iNPH-associated genes in the choroid plexus and ventricular ependyma of the mouse brain. Both light and pseudocolor expression intensity images are shown. Arrows identify hybridization signal in the mouse choroid plexus and ependyma. Data are obtained from the Allen Brain Atlas.

Fig. 2.

Expression of iNPH-associated proteins in the ventricular region of the mouse brain. Immunofluorescence micrographs showing immunoreactivity for proteins expressed from iNPH-associated genes in the ependymal layer and subventricular zone of mice. Immunohistochemistry for Cwh43, PRKD1, Rxfp2, Ak9, and Myo7a was performed using cryostat sections from 7-wk-old C57BL6 mice. Cilia were visualized using an antibody directed against acetylated α-tubulin (Ac-tubulin). Nuclei were stained with DAPI. (Scale is 25 µm.)

Fig. 3.

Relationship between proteins from iNPH-associated genes and ependymal cilia. (A) High resolution immunohistochemistry showing localization of SPG11/spatacsin, Ak9, HAVCR1/Kim1, Notch1, and otogelin immunoreactivity in ependymal cells. Cilia were visualized using an acetylated α-tubulin antibody (green). (Scale is 5 µm.) (B) Immunocytochemistry for Rxfp2 or Myo7a (red) in cultured mouse ependymal cells. Cilia were visualized using an acetylated α-tubulin antibody (green). Arrows show areas where iNPH-associated protein immunoreactivity colocalizes with cilia. (Scale is 5 µm.)

Fig. 4.

iNPH-associated AK9 mutations are damaging mutations. (A) Schematic diagram showing domain structure of Ak9 and location of iNPH-associated AK9 mutations. (B) Western blot showing expression of WT Ak9 protein and Ak9 protein harboring iNPH-associated mutations. Expression vectors encoding GFP (control), wild-type human Ak9 (WT), or human Ak9 proteins harboring one of the iNPH-associated AK9 mutations were used to express the encoded control or mutant proteins in a cell-free in vitro transcription and translation system. (C) In vitro kinase assays were performed to measure the conversion of ADP to ATP by recombinant WT Ak9 and Ak9 harboring iNPH-associated mutations. ATP was detected using a spectrophotometric method. Recombinant Ak9 proteins were expressed using equal amounts of cDNA in a cell-free in vitro transcription and translation system.

Fig. 5.

AK9 mutation decreases sperm motility and progression. (A) (Left) Time lapse video microscopy images demonstrating flagellar excursion of mouse sperm obtained from wild-type (WT) and AK9−/− mice. (Middle) Superimposed camera lucida drawings of WT and AK9−/− mouse sperm illustrating flagellar excursion and sperm head forward progression. (Right) Quantification of flagellar motion data shown in Left and Middle panels. (P < 0.01, unpaired t test). (B) (Left) Time lapse video microscopy images demonstrating motility and progression of mouse sperm obtained from WT and AK9−/− mice. (Right) Quantification of sperm progression data shown in the Left panel. (P < 0.001, unpaired t test). (C) Fluorescence and light micrographs showing Ak9 immunoreactivity (red) in WT mouse sperm. (Scale is 5 µm.) (D) Transmission electron micrographs showing the structure of flagella from WT and AK9−/− mouse sperm. (Scale is 0.5 µm.)

Fig. 6.

iNPH-associated AK9 mutation causes a communicating hydrocephalus. (A) T2-weighted axial magnetic resonance (MR) images of mouse brains obtained from wild-type (WT) and AK9−/− mice showing the size of the lateral, third, and fourth ventricles. (Scale is 1 cm.) (B) T2-weighted axial, coronal, and sagittal MR images indicating the presence of CSF within the cerebral aqueduct and fourth ventricle (arrows). (Scale ~1 cm.) (C) Cryostat sections obtained from a wild-type (WT) and an AK9−/− mouse brain. The sections were stained with DAPI and viewed under epifluorescence to identify cell nuclei. Arrows point to the cerebral aqueduct, which is patent. Note the larger size of the aqueduct in the AK9−/− mouse. (Scale ~ 0.5 mm.) (D) Fluorescence micrographs of coronal sections of WT and AK9−/− mouse brains that were harvested 5 min after intraventricular injection of 70 kD fluorescent dextran (red). Note the presence of dextran in the subarachnoid and paravascular spaces. (Scale ~ 0.5 mm.)

Fig. 7.

iNPH-associated AK9 mutation causes age-dependent communicating hydrocephalus and decreases cilia beat frequency. (A) Ventricular volume in the brains of WT and AK9+/− mice was measured at 3 mo and again at 9 mo using T2-weighted MRI. (B) Rotarod data for WT (n = 7), AK9+/− (n = 7), and AK9−/− (n = 5) mice at 3 mo of age. *= P < 0.0023, unpaired t test. (C) Transmission electron micrographs of the mouse brain ependymal surface (Left) and ependymal motile cilia (Right) from AK9−/− mice. (D) Tracings showing high-speed video microscopy measurements of cilia beat frequency in acute ventricular explants obtained from WT and AK9−/− mice.