Dysbindin-1 in dorsolateral prefrontal cortex of schizophrenia cases is reduced in an isoform-specific manner unrelated to dysbindin-1 mRNA expression

Abstract

DTNBP1 (dystrobrevin binding protein 1) remains a top candidate gene in schizophrenia. Reduced expression of this gene and of its encoded protein, dysbindin-1, have been reported in the brains of schizophrenia cases. It has not been established, however, if the protein reductions encompass all dysbindin-1 isoforms or if they are associated with decreased DTNBP1 gene expression. Using a matched pairs design in which each of 28 Caucasian schizophrenia cases was matched in age and sex to a normal Caucasian control, Western blotting of whole-tissue lysates of dorsolateral prefrontal cortex (DLPFC) revealed significant reductions in dysbindin-1C (but not in dysbindin-1A or -1B) in schizophrenia (P = 0.022). These reductions occurred without any significant change in levels of the encoding transcript in the same tissue samples and in the absence of the only DTNBP1 risk haplotype for schizophrenia reported in the USA. Indeed, no significant correlations were found between case-control differences in any dysbindin-1 isoform and the case-control differences in its encoding mRNA. Consequently, the mean 60% decrease in dysbindin-1C observed in 71% of our case-control pairs appears to reflect abnormalities in mRNA translation and/or processes promoting dysbindin-1C degradation (e.g. oxidative stress, phosphorylation and/or ubiquitination). Given the predominantly post-synaptic localization of dysbindin-1C and known post-synaptic effects of dysbindin-1 reductions in the rodent equivalent of the DLPFC, the present findings suggest that decreased dysbindin-1C in the DLPFC may contribute to the cognitive deficits of schizophrenia by promoting NMDA receptor hypofunction in fast-spiking interneurons.

Figure 1.

Exonic–intronic structure of the 16 known or deduced DTNBP1 pre-mRNA transcripts currently listed on AceView. NCBI accession numbers are given for the three reference sequence transcripts. Only transcripts a–e have been found in the DLPFC (R. Straub, personal communication). The boxed areas are exons, and the chevrons (Λ) between them are introns. Unshaded portions of exons are untranslated regions. The black rectangles indicate the locations of targeted RNA sequences of primers used in this study with the abbreviation of the primers (DYS-95, DYS-102, DYS-d and DYS-e) given above them.

Figure 2.

Dysbindin-1 isoforms detected in the DLPFC with the three antibodies used in this study (Oxford PA3111A, UPenn 331 and UPenn 329). (A) Compares the isoforms as characterized by Talbot et al. (4). Numbers below isoforms designate amino acid (aa) sequence location. CCD, coiled-coil domain composed of helices 1 and 2 (H1 and H2) separated by a stutter region (SR); CTR, C-terminus region; DD, the dysbindin domain; LZM, leucine zipper motif (LZM); NTR, amino terminus region; PD, PEST domain. X1 and X2 are simply uncharacterized regions. (B) Shows dysbindin-1A with the location of immunogens for Oxford PA3111A, UPenn 331 and UPenn 329. The immunogen for the Oxford antibody was amino acids 196–352 of mouse dysbindin-1A, which is 352 amino acids in length compared with 351 amino acids in humans. The UPenn antibodies were made to sequences indicated in human dysbindin-1A. (C) Shows the dysbindin-1 isoforms recognized by Oxford PA3111A (1:1000), UPenn 331(1:6000) and UPenn 329 (1:40) in Western blots of whole-tissue lysates of the DLPFC (50 µg per lane) from the same three normal humans.

Figure 3.

Representative Western blots showing relative amounts of dysbindin-1 isoforms in the DLPFC of schizophrenia (S) cases compared with psychiatrically normal (N) controls matched for age and sex. Reference samples (R1 and R2) were run to assess differences across blots due to variations in experimental conditions. Numbers identify which of the 28 matched pairs tested are shown. Twenty micrograms/lane of protein were electrophoresed, transferred to PVDF membrane and probed with anti-dysbindin-1 antibodies with Oxford PA3111 in (A and C) and with UPenn 331 in (B). Blots were processed for β-actin (D) or MemCode staining (E) to control for variations in protein levels due to sample degradation (during tissue storage or lysate preparation), gel loading and/or efficiency in membrane transfer.

Figure 4.

Levels of dysbindin-1A protein (A) and estimate of its mRNA levels (B) in the DLPFC of the 28 schizophrenia cases compared to their matched controls. For each of these pairs, the amount of the protein in the schizophrenia case to that in its matched control is expressed as a ratio shown on the Y-axis. The ratio is actually the mean of ratios calculated in two separate experiments on the same cases. Mean ratios were log transformed as explained in Data analysis section. Three bars are shown for each of the 28 case–control pairs in (A): one for the ratio calculated with raw data (filled bar), another calculated with data normalized to β-actin levels (striped bar) and a third calculated with data normalized with MemCode results (open bar). The order in which case–control data are displayed in (A) simply reflects rank ordering of the 28 pairs with increasingly positive ratios. In (B), however, the order in which case–control data are displayed is identical to the sequence of case–control pairs shown in (A) to facilitate comparison of protein and gene expression ratios for each pair. The mRNA data were normalized to expression of the housekeeping genes B2M, GAPDH and HPRT. Note the clear lack of correspondence between case–control ratios for dysbindin-1A and its transcript.

Figure 5.

Levels of dysbindin-1B protein (A) and its mRNA levels (B) in the DLPFC of schizophrenia cases compared to matched controls (see caption to Fig. 4 for further explanation). Note the clear lack of correspondence between case–control ratios for dysbindin-1B and its transcript.

Figure 6.

Levels of dysbindin-1C protein (A) and its mRNA levels (B) in the DLPFC of schizophrenia cases compared to matched controls (see caption to Fig. 4 for further explanation). Note the clear lack of correspondence between case–control ratios for dysbindin-1C and its transcript.

Figure 7.

Relative expression levels of targeted DTNBP1 transcripts in DLPFC of schizophrenia (S) cases and matched normal (N) controls as determined with qRT–PCR using primer sequences specified in Table 2. For each of the four different transcripts, the data points (open circles) are normalized values divided by the mean expression level for that transcript on all samples (S and N) so that data for all four transcripts could be plotted on the same scale despite much higher levels of dysbindin-1A than dysbindin-1B or -1C transcripts.