Decreased mRNA expression of netrin-G1 and netrin-G2 in the temporal lobe in schizophrenia and bipolar disorder

Abstract

The membrane-bound axon guidance molecules netrin-G1 (NTNG1) and netrin-G2 (NTNG2) play a role in synaptic formation and maintenance. Non-coding single nucleotide polymorphisms (SNPs) in both genes have been reported to be associated with schizophrenia. The main aim of this study was to determine if NTNG1 and NTNG2 mRNA expression is altered in schizophrenia or bipolar disorder, and/or influenced by disease-associated SNPs. NTNG1 and NTNG2 mRNAs were examined in the medial and inferior temporal lobe using in situ hybridization and RT-PCR in the Stanley Medical Research Institute array collection, and in rat hippocampus during development and after antipsychotic administration. NTNG1 mRNA isoforms were also examined during human brain development. For NTNG1, the G1c isoform was reduced in bipolar disorder and with a similar trend in schizophrenia; expression of four other NTNG1 isoforms was unchanged. In both schizophrenia and bipolar disorder, NTNG2 mRNA was reduced in CA3, with reductions also found in CA4 and perirhinal cortex in bipolar disorder. The SNPs did not affect NTNG1 or NTNG2 mRNA expression. Both NTNG1 and NTNG2 mRNAs were developmentally regulated, and were unaltered by haloperidol, but NTNG2 mRNA was modestly increased by clozapine. These data implicate NTNG1 and NTNG2 in the pathophysiology of schizophrenia and bipolar disorder, but do not support the hypothesis that altered mRNA expression is the mechanism by which genetic variation of NTNG1 or NTNG2 may confer disease susceptibility.

Figure 1

A: Structure of the human NTNG1 gene, showing the alternatively spliced exons 6-9, and the position of SNP rs1373336. The SNP tags a haplotype block that extends as far upstream as the third intron. The forward primer used to amplify G1 isoforms was located in exon 4, with the reverse primer, and the ISHH probe, in exon 10. B: NTNG1 splice isoforms expressed in human brain. C: Structure of the human NTNG2 gene and location of SNP rs1105684. The ISHH probe spanned the exon 7/8 boundary, and is predicted to detect all NTNG2 splice variants based upon the known mouse transcripts.

Figure 2

Regional distribution of (A) NTNG1 and (B) NTNG2 mRNAs in the temporal lobe of representative control subjects. CA: cornu ammonis; DG: dentate gyrus; ITC: inferior temporal cortex; SUB: subiculum: PHG: parahippocampal gyrus; PRC: perirhinal cortex. Although NTNG2 mRNA was observed in the PHG of some subjects (as seen here), its variable expression between individuals precluded quantitative analyses.

Figure 3

Cellular expression of (A-C, E) NTNG1 and (D, F) NTNG2 mRNAs in (A-D) human temporal lobe and (E, F) P42 rat hippocampal formation. A: NTNG1 mRNA is expressed by occasional putative hippocampal interneurons (arrow), as observed here in the stratum oriens of CA3. B: NTNG1 mRNA over pyramidal neurons in the subiculum. C: NTNG1 mRNA expression by layer 3 perirhinal cortical neurons. D: NTNG2 mRNA in the perirhinal cortex is localized over deep layer 5/6 neurons. E: NTNG1 mRNA is expressed by granule cells of the rat DG, and by putative interneurons (arrows), but is not reliably detected over pyramidal neurons (asterisk). F: NTNG2 mRNA is expressed by granule cells of the rat DG, but is not reliably detected over putative hippocampal interneurons (arrow). Bars in (D) and (E) = 30 μm. Abbreviations as in Figure 2.

Figure 4

Representative autoradiographic images demonstrating the regional distribution of (A-D) NTNG1 and (E-J) NTNG2 mRNAs in the developing rat hippocampus. Arrow in E indicates the location of the developing hippocampus. NTNG1 mRNA was not detected in the hippocampus at E19 (not shown), and was first detected in CA1 at P3 (not shown). At P7 (A) NTNG1 mRNA was also detected in the DG. Note that NTNG1 mRNA expression in the DG increases with age, whilst that in CA1 decreases. As previously reported (Nakashiba et al, 2000; Yin et al., 2002) NTNG1 mRNA is strongly expressed in the thalamus. NTNG2 mRNA was detected from E19 onwards and is robustly expressed in most hippocampal subfields. THAL: thalamus. Other abbreviations as in Figure 2.

Figure 5

Relative expression of NTNG1 isoform mRNAs during human brain development. Four isoforms (G1c, G1d, G1e, and G1m) were reliably detected in fetal brain and adult hippocampus and temporal cortex. The relative abundance (expressed as a percentage of the sum of all four mRNAs quantified) of G1c increased, and that of G1d decreased, between fetal and adult brain.

Figure 6

Examples of gel-like images produced by the Agilent 2100 bioanalyzer for semi-quantitative analysis of NTNG1 mRNA using RT-PCR, showing a representative control subject (C), a subject with schizophrenia (S), and a bipolar disorder subject (BP). Bands of the predicted size (ladder on left) corresponding to the G1a, G1c, G1d, G1e and G1m isoforms were detected.

Figure 7

Quantitative analyses of NTNG1 and NTNG2 mRNA expression in the temporal lobe in controls (squares), subjects with schizophrenia (triangles), and subjects with bipolar disorder (circles). A: G1c isoform of NTNG1 mRNA in homogenized temporal cortex tissue. One subject with schizophrenia was omitted from the G1c study due to amplification failure. B-F: NTNG2 mRNA in subfields of the temporal lobe.