MARCKS-dependent mucin clearance and lipid metabolism in ependymal cells are required for maintenance of forebrain homeostasis during aging

Ependymal cells (ECs) form a barrier responsible for selective movement of fluids and molecules between the cerebrospinal fluid and the central nervous system. Here, we demonstrate that metabolic and barrier functions in ECs decline significantly during aging in mice. The longevity of these functions in part requires the expression of the myristoylated alanine-rich protein kinase C substrate (MARCKS). Both the expression levels and subcellular localization of MARCKS in ECs are markedly transformed during aging. Conditional deletion of MARCKS in ECs induces intracellular accumulation of mucins, elevated oxidative stress, and lipid droplet buildup. These alterations are concomitant with precocious disruption of ependymal barrier function, which results in the elevation of reactive astrocytes, microglia, and macrophages in the interstitial brain tissue of young mutant mice. Interestingly, similar alterations are observed during normal aging in ECs and the forebrain interstitium. Our findings constitute a conceptually new paradigm in the potential role of ECs in the initiation of various conditions and diseases in the aging brain.


Animals
All animals were maintained at animal facilities at North Carolina State University and Sanford Research according to guidelines from Institutional Animal Care and Use Committees. The following mouse strains were used for visualizing ECs in vivo: FOXJ1:EGFP mice to label ependyma green (Jacquet et al., 2009b)

Clca3 Lentiviral Production
Full-length mouse Clca3 tagged to Yellow Fluorescent Protein (Clca3::YFP; gift of Dr. Lars Mundhenk Freie Universitaet Berlin, Germany) was subcloned into a vector derived from the replication incompetent equine infectious anemia virus (EIAV) which is highly suitable for selective transduction in ependymal cells (Jacquet et al., 2009a). Clca3 lentivirus was produced by triple transfection of HEK293 cells as previously described (Jacquet et al., 2009a).

Acute-and Long-term Ependymal Cultures and Time-lapse Imaging
For acute in vivo experiments the MARCKS::YFP construct (Fang et al., 2013) was electroporated into adult brains (2 months and 2 years old Fc:tdTom mice) following procedures described before (Barnabe-Heider et al., 2008). Briefly, 20 µg of the construct was stereotaxically injected into the lateral ventricle of anesthetized mice; 1 mm lateral and 2.5 mm deep at the Bregma using manually guided Hamilton microliter syringes. The syringe was slowly retracted after 5 minutes of injection and mice were electroporated by delivering five 50 ms pulses of 200 V at 950 ms interval to the lateral surface of the skull. Following electroporation, mice were allowed to recover for 48 hours and their brains were harvested for wholemount cultures as described before .
Wholemounts were live imaged on a glass bottom dish using an air 40x objective and an Olympus confocal microscope equipped with temperature and CO2 control.
PMA was administered to culture media at a final concentration of 200 nM and live imaging was continued.
For long term experiments the EIAV-FOXJ1:Clca3::YFP lentivirus was injected into the lateral ventricles of MARCKS-cKO and control mice at P0. Mice were sacrificed 24 hours post injection, and ECs were cultured as previously described (El Zein et al., 2009;Guirao et al., 2010). The timing of electroporation (P0) was selected as radial glial cells in the ventricular zone begin their differentiation into ECs perinatally (Jacquet et al., 2009b) and are highly suitable for culturing at this age.

Wholemount stimulation, immunoprecipitation and Western Blotting
Mice were sacrificed by avertin overdose and subependymal wholemounts were dissected and either maintained for pharmacological manipulation or fixed and stained for histology as previously described  BSA for 1 hour at room temperature followed by overnight incubation with primary antibody at 4ºC, repeated washes and incubated in secondary antibody. Blots were then exposed to chemiluminescent substrate (BioRad) and imaged. Blots were stripped and reprobed with antibodies using standard procedures.

Histology
Brains harvested from mice were post-fixed overnight in 4% paraformaldehyde, embedded in 3% low melting agarose, and sectioned at 50 µm in the sagittal plane.
Sections were blocked for 1 hour at room temperature with 10% goat serum and 1% Triton X 100 in 1x PBS followed by ON incubation with primary antibodies

Cell counts and fluorescence intensity quantifications
Clca3 distribution within ECs was quantified by designation of the immunolabelling patterns as fibrillary (fibrous ring-like pattern) or punctate (spotty distribution). ECs with either fibrillary or punctate distribution patterns were counted in high resolution confocal Z-stack images obtained using a 60x objective on an Olympus Fluoview FV1000 confocal microscope. 300 Clca3+ ECs were counted per animal and percentages were determined for each pattern of distribution.

Signal intensities for various immunostained markers were quantified in
Image J using gray scale images from individual fluorophore channels in single slices of Z-stack confocal captures. For measurements in individual ECs, images were obtained with a 60x oil objective lens and digitally zoomed at 2x (Olympus

Ependymal barrier function assay
Ependymal wholemounts were harvested from mice and mounted in 0.012 cm 2 aperture tissue cassettes, mounted in Ussing chambers (Physiologic Instruments, Inc, San Diego, CA) for measurement of epithelial permeability in terms of transepithelial electrical resistance (TER) and FITC dextran permeability as previously described (Smith et al., 2010). While on the Ussing chambers, ependymal wholemounts were bathed in oxygenated Ringer solution and maintained at 37°C via a circulating water bath. 10 mM glucose was added to the chamber in contact with the basal (interstitial) side of the wholemount, and was osmotically balanced with 10 mM mannitol on the apical (CSF) chamber (Fig. 5A).
After a 15 minute stabilization period, FITC-dextran (FD4; 2.2 mg/mL, 4.4 kDa; Sigma) was added to the apical reservoir. After another 15 minutes of equilibration, 100 μL samples (in triplicates) were collected from the basal side of wholemounts at 15 minute intervals for 30 minutes and transferred into a 96 well assay plate.
The presence of FD4 was assayed by measurement of fluorescence intensity using an fMax Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA) and concentrations were determined from standard curves generated by serial dilution of FD4. FD4 flux rates were calculated by subtracting the initial FD4 concentration from final FD4 concentration at the end of 30 minute period and presented as FD4 flux rate in mg/min.

Supporting Information References
Barnabe