Absence of Claudin 11 in CNS Myelin Perturbs Behavior and Neurotransmitter Levels in Mice

Neuronal origins of behavioral disorders have been examined for decades to construct frameworks for understanding psychiatric diseases and developing useful therapeutic strategies with clinical application. Despite abundant anecdotal evidence for white matter etiologies, including altered tractography in neuroimaging and diminished oligodendrocyte-specific gene expression in autopsy studies, mechanistic data demonstrating that dysfunctional myelin sheaths can cause behavioral deficits and perturb neurotransmitter biochemistry have not been forthcoming. At least in part, this impasse stems from difficulties in identifying model systems free of degenerative pathology to enable unambiguous assessment of neuron biology and behavior in a background of myelin dysfunction. Herein we examine myelin mutant mice lacking expression of the Claudin11 gene in oligodendrocytes and characterize two behavioral endophenotypes: perturbed auditory processing and reduced anxiety/avoidance. Importantly, these behaviors are associated with increased transmission time along myelinated fibers as well as glutamate and GABA neurotransmitter imbalances in auditory brainstem and amygdala, in the absence of neurodegeneration. Thus, our findings broaden the etiology of neuropsychiatric disease to include dysfunctional myelin, and identify a preclinical model for the development of novel disease-modifying therapies.

Custom microinjection needles 10-20μm in diameter were pulled from glass capillary tubes using a glass electrode puller (Narishige International Inc., East Meadow, NY) and backfilled with 3μl of a 50mg/ml solution containing Alexa488 labeled Dextran (Molecular Probes, Eugene OR). Needles were attached onto a digital micro-pipette (VWR International, Radnor, PA) fastened to a micromanipulator (Narishige International Inc., East Meadow, NY).
Auditory brainstem slices were transferred from the resting dish into a separate 60mm dish containing freshly carbox-perfused aCSF under a stereomicroscope (Leica, Solms, Germany). The tip of the needle was inserted 0.2 -0.8mm below the surface of the tissue sections and 30nl of dye injected into target nuclei or fiber tracts. Dye was injected unilaterally in 1 -3 bolus deposits and either MNTB or LSO connections were analyzed in each tissue slice. Dextran-labeled fibers from cochlea nucleus injections that entered the contralateral MNTB and were analyzed, as were ipsilateral dextran-labeled fibers entering the LSO. Brain slices were perfused with oxygenated aCSF for 4-6hr to allow for dye uptake at the site of injection and transport down the axons. Slices were fixed overnight with fresh 4% paraformaldehyde (PFA; Sigma, St Louis, MO) at 4°C with gentle rotation then washed in PBS for vibratome sectioning.
Dextran dye-labeled coronal mouse brain sections were removed from PBS, blotted dry, placed into a 15 x 15mm cryomold (Electron Microscopy Science, Thermo Fisher) and overlaid with 4% low-melt agarose (Agarose Products, Hernando, MS). Once solidified, molds were removed, the gel excised, trimmed, and superglued to a vibratome chuck. Coronal sections 70μ m thick were cut (Vibratome Series 1000 Plus) and transferred to a 24-well dish (Thermo Fisher) for antibody labeling.
Slices washed 2 x 30min with 0.5% triton X-100 in PBS. Secondary antibodies conjugated with Alexa568 (Molecular Probes) were diluted in blocking solution and added to the sections for 6hr at room temperature with gentle agitation. The secondary antibodies were removed and replaced with a DAPI solution (Sigma) diluted in PBS plus 0.5% triton X-100 for 10min. Finally, sections were washed 2 x 30min with 0.5% triton X-100 in PBS. Sections were floated in PBS and mounted on slides with Vectashield (Vector Laboratories).

Ox3ZG construct
To generate the Ox3ZG construct, a genomic fragment from a 129 Sv/Ev library containing the 3' end of the mouse Cldn11 gene (Phage #2, Fig.2A 3 ) was restriction endonuclease-digested with Not I, sub-cloned into a pUC2.2 plasmid 4 and further restriction-digested with Fsp I and Not I to remove an 11 kilobase fragment comprising the 3' end of intron 2, exon 3 and 5 kilobases of downstream sequence. This fragment was blunt-end sub-cloned into the Nae I site of a transgene expression cassette containing a 158 base pair thymidine kinase minimal promoter immediately upstream of the lacZ coding region and a genomic fragment from the human beta-globin gene containing exon 2 through exon 3 5 . The resulting pUC2.2-Ox3ZG plasmid was digested with Not I and a 16 kilobase fragment purified from agarose for male pronuclear injection in single cell 0.5 day old mouse embryos. Mice were genotyped using a standard PCR protocol with the following primer pair in mouse Cldn11 Transient transgenic mice Pronuclear injections were performed according to institutional protocols at the University of Michigan Transgenic Animal Model core facility. Founder mice from independent Ox3ZG transgenic lines were perfused for 15min using 2% paraformaldehyde, 2mM MgCl 2 in 0.1M PIPES buffer, pH6.9, according to previously established protocols 5 . Brains were dissected, cut with a razor blade along the sagittal midline and permeabilized overnight with 0.01% deoxycholate (Sigma), 0.02% NP-40 (Sigma), and 2mM MgCl 2 in PBS, then incubated overnight at 37°C in permeabilization solution containing 17.5mM K 3 Fe(CN) 6 , 17.5mM K 4 Fe(CN) 6 , and 1mg/ml X-gal (5-bromo-4-chloro-3-hydroxyindole; Thermo Fisher Scientific). Whole mount stained brains were photographed using a Nikon SMZ1500 dissecting microscope equipped with an HR Plan Apo, WD 54 1X lens, and Sony DKC-5000 digital camera.
Northern blotting Whole brains from adult mice were rapidly dissected from decapitated mice and frozen in dry ice for storage at -80°C. Brains were partially thawed in 4M guanidinium chloride, 0.1M mercaptoethanol and homogenized (Tekmar tissuemizer, Tekman Co, Cincinnati, OH) for CsCl 2 purification as previously described 5,7 . Full-length Cldn11 cDNA was used to probe the northern blots.
Tail suspension test Mice were suspended in air by taping their tail to a horizontal rod which was elevated 12 inches above a table surface. Before applying the tape, a 2.5cm length of polypropylene tubing (loosely fitting) was placed over the tail to prevent mice from grabbing and crawling up their tail during testing. Mice were suspended for 6min and video recorded for subsequent analysis, where the total times spent immobile versus moving were determined. Supplementary Fig. S3 -ABR wave V latency is not rescued by the Tg(Cldn11)605Gow transgene The latency of wave V of the ABR is commonly used to estimate the relative conduction velocity along myelinated fibers in the auditory pathway of a number of mammalian species including humans, but the transmission time of auditory signals in the brainstem is measured directly in the current study. The Tg(Cldn11)605Gow transgene rescues hearing threshold deficits and low endocochlear potential in Cldn11-null mice ( Figure S2) associated with the loss of claudin 11 expression in the basal cells of the stria vascularis 6 . However, the transgene is not expressed in oligodendrocytes of Cldn11 -/-::Tg +/mice and the transmission time defect persists in these animals 3,9,10 . One-way ANOVA F (7,33) = 12.04; p < .0001; ***, p < .001; ****, p < .0001 for Bonferroni's multiple comparisons tests.