ORIGINAL ARTICLE ONLINE FIRST Molecular and Genetic Evidence for Abnormalities in the Nodes of Ranvier in Schizophrenia Panos Roussos, MD, PhD; Pavel Katsel, PhD; Kenneth L. Davis, MD; Panos Bitsios, MD, PhD; Stella G. Giakoumaki, PhD; Jigar Jogia, PhD; Kinga Rozsnyai, MSc; David Collier, PhD; Sophia Frangou, MD, PhD; Larry J. Siever, MD; Vahram Haroutunian, PhD Context: Genetic, neuroimaging, and molecular neu- white men and women participated in the cognitive robiological evidence support the hypothesis that the dis- (n=513) and neuroimaging (n=52) studies. connectivity syndrome in schizophrenia (SZ) could arise from failures of saltatory conduction and abnormalities Main Outcome Measures: The mRNA and protein at the nodes of Ranvier (NOR) interface where myelin levels in postmortem brain samples, genetic association and axons interact. with schizophrenia, cognitive performance, and blood oxygenation level–dependent functional magnetic reso- Objective: To identify abnormalities in the expression nance imaging. of oligodendroglial genes and proteins that participate in the formation, maintenance, and integrity of the NOR Results: The mRNA expression of multiple NOR genes in SZ. was decreased in schizophrenia. The ANK3 rs9804190 C allele was associated with lower ANK3 mRNA expres- Design: The messenger RNA (mRNA) expression lev- sion levels, higher risk for SZ in the case-control cohort, els of multiple NOR genes were quantified in 2 indepen- and poorer working memory and executive function per- dent postmortem brain cohorts of individuals with SZ, formance and increased prefrontal activation during a and generalizability to protein expression was con- firmed. The effect of the ANK3 genotype on the mRNA working memory task in healthy individuals. expression level was tested in postmortem human brain. Case-control analysis tested the association of the ANK3 Conclusions: These results point to abnormalities in the genotype with SZ. The ANK3 genotype’s influence on cog- expression of genes and protein associated with the in- nitive task performance and functional magnetic reso- tegrity of the NOR and suggest them as substrates for the nance imaging activation was tested in 2 independent co- disconnectivity syndrome in SZ. The association of ANK3 horts of healthy individuals. with lower brain mRNA expression levels implicates a molecular mechanism for its genetic, clinical, and cog- Setting: Research hospital. nitive associations with SZ. Patients: Postmortem samples from patients with SZ and Arch Gen Psychiatry. 2012;69(1):7-15. healthy controls were used for the brain expression study Published online September 5, 2011. (n=46) and the case-control analysis (n=272). Healthy doi:10.1001/archgenpsychiatry.2011.110 URING THE PAST DECADE, (NOR).4 The NOR span the entire length multiple lines of evi- of myelinated axons and comprise my- dence (gene and protein elin sheath–free segments where ion (so- expression, genetic asso- dium and potassium) exchange can take ciation, neuroimaging) place, propagating action potentials from Dhave suggested that the disconnectivity the axon initial segment down the length syndrome thought to underlie the neuro- of the axon to synaptic terminals. In the biology of schizophrenia (SZ) is in part due central nervous system, NOR are formed to abnormalities in myelination and oli- and maintained through reciprocal inter- godendroglial function.1-3 Efficient and fast actions between neurons and oligoden- neurotransmission is dependent on salta- drocytes.5 Multiple and specific neuronal tory conduction, which is in turn depen- and oligodendroglial proteins such as an- Author Affiliations are listed at dent on the integrity of myelin sheaths, oli- kyrin G (AnkG) interact to ensure the high the end of this article. godendrocytes, and the nodes of Ranvier concentration (Ͼ1200 voltage-gated so- ARCH GEN PSYCHIATRY/ VOL 69 (NO. 1), JAN 2012 WWW.ARCHGENPSYCHIATRY.COM 7 ©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 dium ion channels/µm2) and anchoring of voltage-gated val, tissue pH, and sex distributions of the subjects are shown sodium ion channels (Nav; Nav1.6 in adults) to the NOR in eTable 1 (http://www.archgenpsychiatry.com). and maintain tight junctions between the axolemma and Gray matter from 14 cortical-frontal (Brodmann area [BA] paranodal myelin loops.5 The AnkG protein belongs to 8, 10, 44, 46, 4), cingulate (BA 23/31, 24/32), parietal (BA 7), a family of scaffolding proteins that interact with sev- temporal (BA 20, 21, 22, 36/28, 38), and occipital (BA 17) brain regions and 3 noncortical brain regions (caudate, hippocam- eral proteins, including the cytoskeletal protein ␤IV spec- 5,6 pus, and putamen) were used for the Affymetrix HG-U133AB trin and Nav channels. The AnkG and Nav subunits GeneChip gene expression analysis (n=17-39) (Affymetrix, also interact in cis with neurofascin 186 and paranodal Santa Clara, California) as described in detail previously.7,8,24 contactin.5,6 Contactin and Caspr interact in trans with Similarly prepared aliquots from the superior temporal gyrus cell adhesion molecules of oligodendroglial paranodal (STG) and primary visual cortex were used in quantitative poly- loops that include the neurofascin 155 isoform and Tag1 merase chain reaction (qPCR) and quantitative Western blot- to ensure the integrity of the myelin-axolemma tight junc- ting analysis in a larger and independent set of brain tissue speci- tion.5,6 Abnormalities in the expression of oligodendrog- mens (eTable 1).25,26 lial and neuronal genes and proteins in SZ could ham- Additional studies for genetic association of ANK3 rs9804190 per saltatory conduction by affecting the formation, were carried out using case-control samples from the Brain Bank. For analytical purposes, only white American individuals with maintenance, and integrity of the NOR and contribute 4 Western European ancestry (SZ, n=208; comparison control, to the hypothesized disconnectivity syndrome. n=64) were used for the clinical genetic investigation of the The current studies were undertaken to advance the ANK3 rs9804190 polymorphism. observation of myelin and oligodendrocyte gene and pro- tein expression abnormalities in SZ to the functional do- Healthy Individuals main and to the study of genetic association based on di- rect neurobiological observations in the brains of persons Learning on Genetics of Schizophrenia Spectrum Cohort and with SZ with specific gene and protein expression ab- Cognitive Task Performance. We examined the effect of the normalities. Exploratory analysis of data from a large post- ANK3 rs9804190 polymorphism using intermediates pheno- mortem microarray study of multiple brain regions of per- types in 530 individuals from the Learning On Genetics Of 7,8 Schizophrenia Spectrum (LOGOS) cohort. This cohort has been sons with SZ suggested the abnormal expression of many 27 neuron- and oligodendrocyte-enriched genes that en- described in detail previously. code proteins associated with paranodal junctions and 9 British Cohort and Neuroimaging Data. We further explored NOR. We therefore sought to identify the involvement the effect of the ANK3 rs9804190 polymorphism on brain ac- of axoglial interacting genes and proteins in SZ more di- tivity (on functional magnetic resonance imaging [fMRI]) dur- rectly by using postmortem brain tissue from an inde- ing a working memory task. Fifty-two healthy volunteers of white pendent cohort of persons with SZ and unaffected com- British descent were assessed with the Structured Clinical In- parison controls. terview for DSM-IV-TR Axis I Disorders28 and the Family In- ANK3 was among the genes that were abnormally ex- terview for Genetic Studies29 to exclude any personal psychi- pressed in independent gene and protein expression stud- atric history or family history (up to second-degree relatives) ies. Because recent genome-wide association studies have of SZ or affective disorders. Participants were further screened implicated ANK3 in bipolar disorder (BD)10-13 and SZ14 to exclude past, current, and hereditary medical disorders, and because several studies have suggested commonali- DSM-IV lifetime drug or alcohol dependence and drug or al- cohol abuse in the preceding 6 months, and contraindications ties in the neurobiology,15-17 etiology, and genetic asso- 12,13,18,19 to MRI. For all participants, an estimate of the current full- ciations between SZ and BD, we also sought to de- scale IQ was obtained on the day of scanning using the Wechs- termine whether ANK3 genetic variants affected the ler Adult Intelligence Scale–Revised,30 while handedness was expression of ANK3 in the same SZ and control brain based on self-report. Written informed consent was obtained specimens and in a larger postmortem collection of ra- from all subjects before study participation. The study was ap- cially homogeneous cases and controls (n=272). The in- proved by the Joint Ethics Committee of the Institute of Psy- fluence of the SZ risk–associated ANK3 polymorphism chiatry and the South London and Maudsley NHS Foundation on cognitive domains of significance to SZ and on brain Trust. activity during performance of a working memory task was also examined in healthy controls (n=513 and n=52, LONG-TERM HALOPERIDOL STUDIES respectively). IN RATS To assess the effects of neuroleptic exposure on the expres- METHODS sion of selected NOR genes of interest, groups of 8 male Sprague- Dawley rats (6-8 months of age) received daily subcutaneous