Several lines of evidence indicate that oligodendrocytes and myelin are disturbed in schizophrenia (Davis et al., 2003; Segal et al., 2007). However, the relationship of these alterations to the pathogenesis of schizophrenia is still unclear. A recent paper by Budel et al. proposes one possible link between oligodendrocyte and myelin pathology and schizophrenia pathogenesis. The gene for Nogo-66 receptor 1 (RTN4R) is located within the 22q11.2 locus where a hemizygous microdeletion (1.5 Mb) occurs at a frequency of one in 5,000. Twenty to 30 percent of individuals with the deletion develop schizophrenia. Several candidate genes for the schizophrenia phenotype within this locus have been characterized for genetic association, and common variants of the Nogo-66 receptor 1 gene have shown association in one study (Liu et al., 2002), but replication studies have not confirmed the findings using different cohorts (  Read more

Several lines of evidence indicate that oligodendrocytes and myelin are disturbed in schizophrenia (Davis et al., 2003; Segal et al., 2007). However, the relationship of these alterations to the pathogenesis of schizophrenia is still unclear. A recent paper by Budel et al. proposes one possible link between oligodendrocyte and myelin pathology and schizophrenia pathogenesis. The gene for Nogo-66 receptor 1 (RTN4R) is located within the 22q11.2 locus where a hemizygous microdeletion (1.5 Mb) occurs at a frequency of one in 5,000. Twenty to 30 percent of individuals with the deletion develop schizophrenia. Several candidate genes for the schizophrenia phenotype within this locus have been characterized for genetic association, and common variants of the Nogo-66 receptor 1 gene have shown association in one study (Liu et al., 2002), but replication studies have not confirmed the findings using different cohorts (Meng et al., 2007; Hsu et al., 2007). Rare variants for the Nogo-66 receptor 1 gene not found in controls have also been identified in schizophrenia cases (Hsu et al., 2007; Sinibaldi et al., 2004), but their functional or pathological association with schizophrenia has not been demonstrated.

Budel et al. first performed a genetic association study of common variants of Nogo-66 receptor 1 gene and confirmed previous findings in their independent cohort. Because these are intronic variants whose effects on Nogo-66 receptor 1 are not clear, they also searched for rare variants of the gene by direct sequencing of 542 DNA samples from individuals with schizophrenia. They found four previously unreported rare variants that are non-synonymous, giving a rate of non-synonymous rare variants of ~1 percent (eight in 870). These researchers also sequenced 650 control DNA samples and found eight rare non-synonymous variants, which together with previous other studies makes the total number of sequenced control samples 1,250. The overall incidence of coding region variants in controls and schizophrenia does not differ between the two groups. However, a bioinformatic analysis suggested that four out of eight rare variants found in schizophrenia are potentially detrimental to protein function, whereas none of eight rare variants found in controls are, indicating a higher incidence of potentially deleterious variations in the Nogo-66 receptor 1 gene in schizophrenia.

Nogo-66 is a myelin-associated outgrowth inhibitor that is responsible for the inhibition of regeneration of CNS axons. It binds to Nogo-66 receptor 1 which induces repulsive responses from neurons. On this basis, Budel et al. investigated whether these rare variants found in schizophrenia are functional. They found that both the R377Q and R377W variants in the signaling domain could not induce Nogo-66-mediated repulsive response from neurons when they were introduced into neurons that do not express the Nogo receptor. Interestingly, these variants could suppress the repulsive response from neurons that express endogenous Nogo-66 receptor 1, suggesting that they could work as dominant-negative forms. They also showed that Nogo-66 binding domain variants R119W and R196H both showed reduced binding to Nogo-66. Their results demonstrate that some of the rare variants found in schizophrenia (and not in controls) are functional variants, some of which may work as dominant negatives.

They further analyzed Nogo-66 receptor 1 knockout mice for behavioral characteristics relevant to schizophrenia. They found that Nogo-66 receptor 1 knockout mice show impairment in spatial working memory, a promising endophenotype in schizophrenia. This was specific to working memory as no deficits in the radial arm water maze test or passive avoidance test were observed. Nogo-66 receptor 1 knockout mice did not, however, show any changes in PPI, another important phenotype relevant for schizophrenia mouse models.

The authors had shown previously that Nogo-66 receptor-mediated axon growth inhibition is crucial for formation of ocular dominance in the visual cortex in mice, suggesting myelin involvement in brain wiring refinement and restriction of neuronal plasticity (McGee et al., 2005). The prefrontal cortex—believed to be impaired in schizophrenia—completes its myelination during late adolescence and early adulthood, when symptoms of schizophrenia emerge (Benes, 1989). Therefore, their study supports the idea that abnormal myelination may be a risk factor for schizophrenia. Interestingly, another Nogo receptor, pirB, has been identified (Atwal et al., 2008), and PirB is also shown to be involved in ocular dominance formation in the visual cortex (Syken et al., 2006). It would be interesting to look into PirB involvement in myelination and oligodendrocyte differentiation as well as in schizophrenia.

The Budel et al. study leaves a number of issues open. First, the incidence of each rare variant is one in 870 in schizophrenia, and to prove that they are not found in controls would require screening in a much larger number of controls for a reliable genetic study. This is always the problem when we characterize rare variants in association with disorders such as schizophrenia. Second, Hsu et al. also performed a behavioral analysis of their Nogo-66 receptor 1 knockout mice and did not find the working memory deficits that were seen in this study. They both used T maze-based delayed spatial working memory paradigm, but with subtle difference in their protocols. Also, Budel et al. used very strict inclusion/exclusion criteria of animals for the final testing based on animals’ ability during training sessions. Furthermore, as Hsu et al. suggested, there may be differences in mouse genetic background and age, which is not described in this study; Hsu et al. used a C57Blx129 mixed genetic background. Third, as Budel et al. mentioned, a recent study showed that Nogo-66 receptor 1 has synaptic functions, playing a role in glutamate receptor modulation (Lee et al., 2008), and as such it is distinctly possible that the Nogo-66 receptor 1 involvement in pathogenesis of schizophrenia is not related to myelin, but to synaptic deficits. Nevertheless, this study clearly demonstrated that rare variants in Nogo-66 receptor 1 found in individuals with schizophrenia show defects in myelin-mediated neuronal function, which could explain in part the working memory deficits observed in Nogo-66 receptor 1 knockout mice, and may provide a possible causal link between defects in oligodendrocyte function, myelination, and pathogenesis of schizophrenia.

References:

Davis, K.L., et al. (2003) White matter changes in schizophrenia: evidence for myelin-related dysfunction. Arch Gen Psychiatry 60, 443-456. Abstract

Segal, D., et al. (2007) Oligodendrocyte pathophysiology: a new view of schizophrenia. Int J Neuropsychopharmacol 10, 503-511. Abstract

Liu, H., et al. (2002) Genetic variation in the 22q11 locus and susceptibility to schizophrenia. Proc Natl Acad Sci U S A 99, 16859-16864. Abstract

Meng, J., et al. (2007) No association between the genetic polymorphisms in the RTN4R gene and schizophrenia in the Chinese population. J Neural Transm 114, 249-254. Abstract

Hsu, R., et al. (2007) Nogo Receptor 1 (RTN4R) as a candidate gene for schizophrenia: analysis using human and mouse genetic approaches. PLoS ONE 2, e1234. Abstract

Sinibaldi, L., et al. (2004) Mutations of the Nogo-66 receptor (RTN4R) gene in schizophrenia. Hum Mutat 24, 534-535. Abstract

McGee, A.W., et al. (2005) Experience-driven plasticity of visual cortex limited by myelin and Nogo receptor. Science 309, 2222-2226. Abstract Abstract

Atwal, J.K., et al. (2008) PirB is a functional receptor for myelin inhibitors of axonal regeneration. Science 322, 967-970. Abstract

Syken, J., et al. (2006) PirB restricts ocular-dominance plasticity in visual cortex. Science 313, 1795-1800. Abstract

Lee, H., et al. (2008) Synaptic function for the Nogo-66 receptor NgR1: regulation of dendritic spine morphology and activity-dependent synaptic strength. J Neurosci 28, 2753-2765. Abstract

Individuals with hemizygous microdeletions at the 22q11.2 locus display a range of cognitive and behavioral deficits, and compared to the general population these individuals have a greatly increased risk of developing schizophrenia (Karayiorgou et al., 1995). A number of candidate schizophrenia susceptibility genes have been identified within the 22q11.2 region (Mukai et al., 2004; Paterlini et al., 2005; Paylor et al., 2006; Stark et al., 2008). In our paper (Hsu et al., 2007), we evaluated RTN4R (NgR1), one of the genes in the 22q11.2 region, as a schizophrenia susceptibility gene using a variety of approaches including human association analyses as well as mouse behavioral and anatomical assays. We evaluated common RTN4R variants in a large Afrikaner family sample and found RTN4R polymorphisms which...  Read more

Individuals with hemizygous microdeletions at the 22q11.2 locus display a range of cognitive and behavioral deficits, and compared to the general population these individuals have a greatly increased risk of developing schizophrenia (Karayiorgou et al., 1995). A number of candidate schizophrenia susceptibility genes have been identified within the 22q11.2 region (Mukai et al., 2004; Paterlini et al., 2005; Paylor et al., 2006; Stark et al., 2008). In our paper (Hsu et al., 2007), we evaluated RTN4R (NgR1), one of the genes in the 22q11.2 region, as a schizophrenia susceptibility gene using a variety of approaches including human association analyses as well as mouse behavioral and anatomical assays. We evaluated common RTN4R variants in a large Afrikaner family sample and found RTN4R polymorphisms which showed weak sex-specific association with schizophrenia. We also sequenced for rare RTN4R coding variants in an independent sample and identified two novel nonconservative RTN4R coding variants that were found in individuals with schizophrenia but not in a control population. Finally, we observed that Rtn4r-deficient mice had locomotory defects but showed normal behavior in a panel of schizophrenia-related tasks, including working memory.

In a more recently published study, Budel et al. performed similar experiments using independent schizophrenia and control populations. This group identified several nonconservative RTN4R coding variants in schizophrenia patients consistent with previous studies (Sinibaldi et al., 2004; Hsu et al., 2007); as with our study, the screening of a large number of additional patients and controls would be necessary to demonstrate an unequivocal association with schizophrenia. However, the authors provided functional evidence which indicates that some of these variants fail to transduce myelin-derived inhibitory signals in vitro.

Budel and colleagues also evaluated an independently-generated Rtn4r-deficient mouse strain for schizophrenia-related endophenotypes. As was previously reported (Kim et al., 2004), these mice exhibited locomotory deficits including hypoactivity in an open field test, similar to observations by our own group. Interestingly, both our group and the Budel group measured spatial working memory using the delayed alternation T maze assay, but with differing results. While we found no significant differences, Budel et al. reported a deficit in the Rtn4r-deficient mice. There are a number of issues that are raised by this discrepancy as well as several possible explanations.

First, differences in the assay protocols could account for the discrepancy. In our experiments, both the acquisition and retention of working memory were sequentially assayed following the shaping (training) period, whereas Budel et al. averaged data across the 30 days following training, which does not clearly distinguish between the acquisition of the task and the retention of working memory. In addition, Budel et al. used a 2.5 second delay between trials, which would place a smaller load on working memory compared to the 5 and 20 second delays used in our experiments.

Second, the design of the targeting constructs could potentially have an influence on behavior. In our study, we chose a self-excisable selection cassette containing the neo selection marker (Bunting et al., 1999). Excision of the neo gene following germline transmission ensures that any observed phenotype is due to the deletion rather than any long-range transcriptional effects of the selection cassette (Olson et al., 1996). This is particularly important in the case of RTN4R given that the RTN4R gene lies adjacent to ZDHHC8 and PRODH, two other genes in the 22q11.2 region which have been implicated in schizophrenia (Paterlini et al., 2005; Mukai et al., 2008).

Finally, we conducted our experiments in wild-type, heterozygous, and homozygous Rtn4r knockout animals, whereas Budel et al., using smaller cohorts, reported results from only wild-type and homozygous animals in the T maze assay. In the radial-arm water maze, on the other hand, Budel et al. included heterozygotes but omitted the wild-type controls. Comparisons between all three genotypes—homozygous, heterozygous, and wild-type—are particularly relevant in this context given that schizophrenia is strongly associated with a hemizygous deletion of the 22q11.2 region. Furthermore, Budel et al. used pure strain mice for the T maze assay while mixed background mice were used for the water maze; it is unclear to what degree strain effects could play a role (Crawley, 1996). The use of accurate and reliable mouse models is invaluable in analyzing the effect of genetic loci predisposing complex psychiatric disorders (Arguello and Gogos, 2006; Kvajo et al., 2008), and the results discussed here highlight some of the caveats that must be taken into account when interpreting the effect of single gene deletions on complex behaviors such as working memory, particularly when evaluated as an endophenotype of schizophrenia.

References:

Arguello PA, Gogos JA. Modeling madness in mice: one piece at a time. Neuron. 2006. 52(1):179-96. Abstract

Budel S, Padukkavidana T, Liu BP, et al. Genetic variants of Nogo-66 receptor with possible association to schizophrenia block myelin inhibition of axon growth. J Neurosci. 2008. 28(49):13161-72. Abstract

Bunting M, Bernstein KE, Greer JM, et al. Targeting genes for self-excision in the germ line. Genes Dev. 1999. 13(12):1524-8. Abstract

Crawley, JN. Unusual behavioral phenotypes of inbred mouse strains. Trends Neurosci. 1996. 19(5):181-2. Abstract

Hsu R, Woodroffe A, Lai WS, et al. Nogo Receptor 1 (RTN4R) as a candidate gene for schizophrenia: analysis using human and mouse genetic approaches. PLoS ONE. 2007. 2(11):e1234. Abstract

Karayiorgou M, Morris MA, Morrow B, et al. Schizophrenia susceptibility associated with interstitial deletions of chromosome 22q11. Proc Natl Acad Sci U S A. 1995. 92(17):7612-6. Abstract

Kim JE, Liu BP, Park JH, et al. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron. 2004. 44(3):439-51. Abstract

Kvajo M, McKellar H, Arguello PA, et al. A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition. Proc Natl Acad Sci U S A. 2008. 105(19):7076-81. Abstract

Mukai J, Dhilla A, Drew LJ, et al. Palmitoylation-dependent neurodevelopmental deficits in a mouse model of 22q11 microdeletion. Nat Neurosci. 2008. 11(11):1302-10. Abstract

Mukai J, Liu H, Burt RA, et al. Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia. Nat Genet. 2004. 36(7):725-31. Abstract

Olson EN, Arnold HH, Rigby PW, et al. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell. 1996. 85(1):1-4. Abstract

Paterlini M, Zakharenko SS, Lai WS. Transcriptional and behavioral interaction between 22q11.2 orthologs modulates schizophrenia-related phenotypes in mice. Nat Neurosci. 2005. 8(11):1586-94. Abstract

Paylor R, Glaser B, Mupo A, et al. Tbx1 haploinsufficiency is linked to behavioral disorders in mice and humans: implications for 22q11 deletion syndrome. Proceedings of the National Academy of Sciences of the United States of America. 2006. 103(20):7729-34. Abstract

Sinibaldi L, De Luca A, Bellacchio E, et al. Mutations of the Nogo-66 receptor (RTN4R) gene in schizophrenia. Hum Mutat. 2004. 24(6):534-5. Abstract

Stark KL, Xu B, Bagchi A, et al. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat Genet. 2008. 40(6):751-60. Abstract

This is a thorough and generally well-written manuscript that provides further evidence to the hypothesis that schizophrenia may be viewed as a disconnectivity syndrome (Frith, 1996; Davis et al., 2003) due to disturbances in myelination.

Even though the authors examined a large sample consisting of 3 different populations (Caucasians, African-Americans and Chinese Han trio sample), they do not provide details regarding the age-ratio of these populations, nor do they report the treatment of these patients. Hence, there is a growing body of evidence of age-related changes in the human brain (Allen et al., 2005).We consider that the authors of this study fail to investigate of how the effects of age are expressed. It can not be ruled out whether there is any effect of the medication in the observed results. Even though medication is not implicated in the observed alterations in gene expression in schizophrenia in several studies (  Read more

This is a thorough and generally well-written manuscript that provides further evidence to the hypothesis that schizophrenia may be viewed as a disconnectivity syndrome (Frith, 1996; Davis et al., 2003) due to disturbances in myelination.

Even though the authors examined a large sample consisting of 3 different populations (Caucasians, African-Americans and Chinese Han trio sample), they do not provide details regarding the age-ratio of these populations, nor do they report the treatment of these patients. Hence, there is a growing body of evidence of age-related changes in the human brain (Allen et al., 2005).We consider that the authors of this study fail to investigate of how the effects of age are expressed. It can not be ruled out whether there is any effect of the medication in the observed results. Even though medication is not implicated in the observed alterations in gene expression in schizophrenia in several studies (Hakak et al., 2001; Pongrac et al., 2002), the possibility that the expression levels of some genes may be modulated by medication (Pongrac et al., 2002) cannot be disregarded. Ultimately, it is essential to take into consideration that schizophrenia is a syndrome, so less broad diagnostic categories are crucial. These issues must be investigated more intensely, in order to obtain statistically sound results.

Nonetheless, Budel et al. present evidence that there is a positive association of NGR variation in schizophrenic patients which may be relevant to the pathophysiology of the disorder. Their results support the hypothesis that oligodendroglial dysfunction with subsequent abnormalities in myelin maintenance and repair contribute to the schizophrenic syndrome (Davis et al., 2003). Therefore they shed more light on the aetiopathogenesis of schizophrenia and the understanding of its seemingly disparate genetic aspects.

References:

Frith C. Neuropsychology of schizophrenia, what are the implications of intellectual and experiential abnormalities for the neurobiology of schizophrenia? Br Med Bull 1996; 52: 618-626. Abstract

Davis KL, Stewart DG, Friedman JI, Buchsbaum M, Harvey PD, Hof PR et al. White matter changes in schizophrenia: evidence for myelin-related dysfunction. Arch Gen Psychiatry 2003; 60: 443–456. Abstract

Allen JS, Bruss J, Brown CK, Damasio H et al. Normal neuroanatomical variation due to age: The major lobes and a parcellation of the temporal region. Neurobiology of Aging 2005; 26: 1245–1260. Abstract

Hakak Y, Walker JR, Li C, Wong WH, Davis KL, Buxbaum JD et al. Genome-wide expression analysis reveals dysregulation of myelination- related genes in chronic schizophrenia. Proc Natl Acad Sci USA 2001; 98: 4746–4751. Abstract

Pongrac J, Middleton FA, Lewis DA, Levitt P, Mirnics K. Gene expression profiling with DNA microarrays: advancing our understanding of psychiatric disorders. Neurochem Res 2002; 27: 1049–1063. Abstract