Common variants of the genes encoding erythropoietin and its receptor modulate cognitive performance in schizophrenia

Abstract

Erythropoietin (EPO) improves cognitive performance in clinical studies and rodent experiments. We hypothesized that an intrinsic role of EPO for cognition exists, with particular relevance in situations of cognitive decline, which is reflected by associations of EPO and EPO receptor (EPOR) genotypes with cognitive functions. To prove this hypothesis, schizophrenic patients (N > 1000) were genotyped for 5' upstream-located gene variants, EPO SNP rs1617640 (T/G) and EPORSTR(GA)(n). Associations of these variants were obtained for cognitive processing speed, fine motor skills and short-term memory readouts, with one particular combination of genotypes superior to all others (p < 0.0001). In an independent healthy control sample (N > 800), these associations were confirmed. A matching preclinical study with mice demonstrated cognitive processing speed and memory enhanced upon transgenic expression of constitutively active EPOR in pyramidal neurons of cortex and hippocampus. We thus predicted that the human genotypes associated with better cognition would reflect gain-of-function effects. Indeed, reporter gene assays and quantitative transcriptional analysis of peripheral blood mononuclear cells showed genotype-dependent EPO/EPOR expression differences. Together, these findings reveal a role of endogenous EPO/EPOR for cognition, at least in schizophrenic patients.

Figure 1

EPO and EPOR genotype analyses in schizophrenic patients of the GRAS data collection and healthy controls (blood donors). (A) Genetic overview of EPO/EPOR including analyzed genetic markers. (B,C) A case control study reveals comparable distribution of EPO SNP genotypes in schizophrenic and healthy control subjects, thus excluding EPO genotypes as risk factors for schizophrenia. (D,E) Case control analysis of EPOR STR (GA)_n repeat lengths shows comparable results for both samples, again excluding a risk constellation of EPOR genotypes. (F) Grouping of genotype combinations with respect to Digit Symbol-Coding test performance uncovers one genotype highly superior to all others: GG&21–35 (lowest) repeat sum. Mean ± standard error of the mean (SEM) given; χ² tests and analysis of covariance (ANCOVA) applied.

Figure 2

Mice with transgenic expression of constitutively active EPOR (cEPOR) driven by the α-calcium/calmodulin-dependent protein kinase II (α-CaMKII) promoter demonstrate highly superior cognitive performance compared with their wild type littermates. (A) Construct used for transgenic expression. (B) Significant reduction of reaction time in the attentional testing part of the 5-Choice Serial Reaction Time Task (5-CSRTT) reflects superior speed of cognitive processing in transgenic mice. (C–F) Transgenic mice perform better in Novel Object Recognition (NOR) (depicted is the number of visits of the new object) and T-maze tests with or without delay, illustrating their supremacy in memory tasks. Exact N numbers of all experiments are given directly in the bars or the line graph; all male mice, 5–8 month old at the time point of testing; mean ± standard error of the mean (SEM) presented; two-way ANOVA for repeated measures and Mann-Whitney U tests applied.

Figure 3

Genotype-dependent EPO/EPOR expression differences using reporter gene assays and PBMCs. (A) Reporter gene constructs: 5′ upstream region of the EPO gene with either G or T at rs1617640 (left) and in addition with the 3′ regulatory region of EPO (right). (B) G at SNP rs1617640 leads to significantly higher basal gene expression than T (baseline control). Addition of the 3′ regulatory region to μmol/L the construct induces downregulation of gene expression. This suppression is stepwise alleviated by increasing doses of CoCl₂ (100 and 400 μmol/L). (C) In all conditions shown in (B), the suppressability (regulability) of gene expression compared with the baseline control (that is, normoxic control condition of B) is highest for the G genotype. (D) EPOR mRNA levels in peripheral blood mononuclear cells (PBMCs) of patients, determined by qRT-PCR and normalized to GAPDH as the housekeeper, show that the lowest EPOR STR (GA)_n repeat length sum is associated with the highest EPOR expression. Gender distribution among the three repeat groups is well balanced (males/all: 24/37, 18/31, 20/30; χ² test p = 0.628). (E) Even when considering the individual EPO mRNA levels (which by themselves do not reveal differences; data not shown) in form of an EPO/EPOR expression ratio, the significant difference between different STR length carriers remains. N = 28–37 per group; mean ± standard error of the mean (SEM) presented; Mann-Whitney U tests applied.