Karen M. J. van Loo

Gene dosage effect on gamma-secretase component Aph-1b in a rat model for neurodevelopmental disorders.

A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Here, we report microarray analyses revealing in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple (neuro)developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregated with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype.

Rats that differentially respond to cocaine differ in their dopaminergic storage capacity of the nucleus accumbens

Cocaine (COC) inhibits the re‐uptake of dopamine. However, the dopamine response to COC also depends on dopamine inside storage vesicles. The aim of this study was to investigate whether rats that differentially respond to COC differ in their dopaminergic storage capacity of the nucleus accumbens. Total and vesicular levels of accumbal dopamine as well as accumbal vesicular monoamine transporter‐2 levels were established in high (HR) and low responders (LR) to novelty rats. Moreover, the effects of reserpine (RES) on the COC‐induced increase of extracellular accumbal dopamine were investigated. HR displayed higher accumbal levels of total and vesicular dopamine than LR. Moreover, HR displayed more accumbal vesicular monoamine transporters‐2 than LR. COC increased extracellular accumbal dopamine more strongly in HR than in LR. A low dose of RES prevented the COC‐induced increase of accumbal dopamine in LR, but not in HR. A higher dose of RES was required to inhibit the COC‐induced increase of accumbal dopamine in HR. These data demonstrate that HR were marked by a larger accumbal dopaminergic storage pool than LR. It is hypothesized that HR are more sensitive to COC than LR, because COC can release more dopamine from accumbal storage vesicles in HR than in LR.

Clock genes and body composition in patients with schizophrenia under treatment with antipsychotic drugs

CONTEXT In the healthy population, several pathways are known to exert an effect on basal metabolic factors. Previous studies have found associations between single nucleotide polymorphisms in clock genes or downstream hormone receptors such as the leptin receptor (LEPR) or glucocorticoid receptor (NR3C1) and obesity in the healthy population, but this association remains to be examined in patients with schizophrenia treated with antipsychotics. OBJECTIVE To assess anthropomorphic parameters in patients taking second-generation antipsychotics (SGA) as a function of nine polymorphisms in three core genes of the clock pathway, and two genes of downstream hormone receptors. METHODS Clinical parameters were evaluated in 261 patients with schizophrenia spectrum disorder. Polymorphisms in LEPR, MC3R, NR3C1, PER2 and SDC3 were genotyped. In order to control for multiple testing, permutation tests were used to generate corrected empirical p-values using the Max(T) procedure in PLINK. RESULTS A significant effect of the rs6196 polymorphism in the NR3C1 on weight (β=-4.18; SE=2.02; p=0.018), BMI (β=-1.88; SE=0.64; p=0.004), waist (β=-5.77; SE=1.75; p=0.001) and waist/hip ratio (β=-0.03; SE=0.012; p=0.009) was found. Permutation tests confirmed the findings for BMI (p=0.037) and waist (p=0.024). Carriers of the G allele consistently displayed better parameters than patients with the wild type allele. A weak effect of rs4949184 in SDC3 on BMI was found, but this did not sustain permutation testing (β=-1.27; SE=0.58; p=0.030, p=0.270 after permutations). CONCLUSION Variations in genes implicated in circadian regulation or its related downstream pathways may be important in the regulation of antropomorphic parameters in patients with schizophrenia during long-term treatment with SGA.

Three-cohort targeted gene screening reveals a non-synonymous TRKA polymorphism associated with schizophrenia.

Schizophrenia is a complex neurodevelopmental disorder that is thought to be induced by an interaction between predisposing genes and environmental stressors. To identify predisposing genetic factors, we performed a targeted (mostly neurodevelopmental) gene approach involving the screening of 396 selected non-synonymous single-nucleotide polymorphisms (SNPs) in three independent Caucasian schizophrenia case-control cohorts (USA, Denmark and Norway). A meta-analysis revealed ten non-synonymous SNPs that were nominally associated with schizophrenia, nine of which have not been previously linked to the disorder. Risk alleles are in TRKA (rs6336), BARD1 (rs28997576), LAMA5 (rs3810548), DKK2 (rs7037102), NOD2 (rs2066844) and RELN (rs2229860), whereas protective alleles are in NOD2 (rs2066845), NRG1 (rs10503929), ADAM7 (rs13259668) and TNR (rs859427). Following correction for multiple testing, the most attractive candidate for further study concerns SNP rs6336 (q=0.12) that causes the substitution of an evolutionarily highly conserved amino acid residue in the kinase domain of the neurodevelopmentally important receptor TRKA. Thus, TRKA signaling may represent a novel susceptibility pathway for schizophrenia.

Reduced Aph-1b expression causes tissue- and substrate-specific changes in γ-secretase activity in rats with a complex phenotype

The γ‐secretase enzyme complex displays intramembrane catalytic activity toward many type I transmembrane proteins, including the Alzheimer‐linked amyloid‐β precursor protein (APP) and the neuregulin receptor ErbB4. Active γ‐secretase is a tetrameric protein complex consisting of presenilin‐1 (or ‐2), nicastrin, PEN‐2, and Aph‐1a (or ‐1b). We have recently discovered that pharmacogenetically bred apomorphine‐susceptible Wistar rats (APO‐SUS) have only one or two copies of the Aph‐1b gene (termed I/I and II/II rats, respectively), whereas their phenotypic counterparts (APO‐UNSUS) have three copies (III/III). As a result, APO‐SUS rats display reduced Aph‐1b expression and a complex phenotype reminiscent of neurodevelopmental disorders. Here we determined in the I/I and III/III rats the γ‐secretase cleavage activity toward the three APP superfamily members, p75 neurotrophin receptor, ErbB4, and neuregulin‐2, and found that the cleavage of only a subset of the substrates was changed. Furthermore, the observed differences were restricted to tissues that normally express relatively high Aph‐1b compared with Aph‐1a levels. Thus, we provide in vivo evidence that subtle alterations in γsecretase subunit composition may lead to a variety of affected (neuro)developmental signaling pathways and, consequently, a complex phenotype.

Dual association of a TRKA polymorphism with schizophrenia.

Objective An interaction between predisposing genes and environmental stressors is thought to underlie the neurodevelopmental disorder schizophrenia. In a targeted gene screening, we previously found that the minor allele of the single nucleotide polymorphism (SNP) rs6336 in the neurotrophic tyrosine kinase receptor 1 (NTRK1/TRKA) gene is associated with schizophrenia as a risk factor. Methods We genotyped the TRKA SNP in a total of eight independent Caucasian schizophrenia case–control groups. Result Remarkably, although in five of the groups a higher frequency of the risk allele was indeed found in the patients compared with the controls, in the three other groups the SNP acted as a protective factor. Conclusion An intriguing possibility is that this dual character of the TRKA SNP is caused by its interaction with endophenotypic and/or epistatic factors.

Identification of Genetic and Epigenetic Variations in a Rat Model for Neurodevelopmental Disorders

A combination of genetic variations, epimutations and environmental factors may be involved in the etiology of complex neurodevelopmental disorders like schizophrenia. To study such disorders, we use apomorphine-unsusceptible (APO-UNSUS) Wistar rats and their phenotypic counterpart apomorphine-susceptible (APO-SUS) rats that display a complex phenotype remarkably similar to that of schizophrenic patients. As the molecular basis of the APO-SUS/UNSUS rat model, we recently identified a genomic rearrangement of the Aph-1b gene. Here, we discovered between the two rat lines differences other than the Aph-1b gene defect, including a remarkable cluster of genetic variations, two variants corresponding to topoisomerase II-based recombination hot spots and an epigenetic (DNA methylation) difference in cerebellum and (hypo)thalamic but not hippocampal genomic DNA. Furthermore, genetic variations were found to correlate with the degree of apomorphine susceptibility in unselected Wistar rats. Together, the results show that a number of genetic and epigenetic differences exist between the APO-SUS and -UNSUS rat genomes, raising the possibility that in addition to the Aph-1b gene defect the newly identified variations may also contribute to the complex APO-SUS phenotype.

Male-specific association between a gamma-secretase polymorphism and premature coronary atherosclerosis

Background Atherosclerosis is a common multifactorial disease resulting from an interaction between susceptibility genes and environmental factors. The causative genes that contribute to atherosclerosis are elusive. Based on recent findings with a Wistar rat model, we speculated that the γ-secretase pathway may be associated with atherosclerosis. Methodology/Principal Findings We have tested for association of premature coronary atherosclerosis with a non-synonymous single-nucleotide polymorphism (SNP) in the γ-secretase component APH1B (Phe217Leu; rs1047552), a SNP previously linked to Alzheimers disease. Analysis of a Dutch Caucasian cohort (780 cases; 1414 controls) showed a higher prevalence of the risk allele in the patients (odds ratio (OR) = 1.35), albeit not statistically different from the control population. Intriguingly, after gender stratification, the difference was significant in males (OR = 1.63; p = 0.033), but not in females (OR = 0.50; p = 0.20). Since Phe217Leu-mutated APH1B showed reduced γ-secretase activity in mouse embryonic fibroblasts, the genetic variation is likely functional. Conclusion/Significance We conclude that, in a male-specific manner, disturbed γ-secretase signalling may play a role in the susceptibility for premature coronary atherosclerosis.

Dopamine susceptibility of APO-SUS rats is not per se coupled to HPA-axis activity

A synergistic relationship is thought to exist between hypothalamic-pituitary-adrenal (HPA) axis activity and dopamine neurotransmission. To test whether a high response to dopamine indeed implies a hyperactive HPA-axis, we here used Wistar rats that were selected twice independently (original and replicate lines) for a high or low susceptibility to the dopamine receptor agonist apomorphine (so-called APO-SUS and APO-UNSUS rats, respectively). The APO-SUS rats from the original line displayed a hyperactive HPA-axis in that higher basal and stress-induced adrenocorticotropic hormone (ACTH) levels, and lower basal free-corticosterone levels were observed than those found in the original APO-UNSUS rats. In contrast, the activity of the HPA-axis in the APO-SUS rats from the replicate line did not differ from that in the replicate APO-UNSUS rats. Thus, in the APO-SUS/APO-UNSUS rat model the level of HPA-axis activity is not necessarily causally linked to dopamine responsiveness, implying that a hyperactive HPA-axis is not a prerequisite for a high dopaminergic response.

Correlation between HIV-1 seropositivity and prevalence of a gamma-secretase polymorphism in two distinct ethnic populations.

Susceptibility for human immunodeficiency virus type 1 (HIV‐1) infection may be influenced by host genetics. Recent findings with a Wistar rat model raised the possibility that the γ‐secretase pathway may be associated with an individuals susceptibility to infection. A functional single‐nucleotide polymorphism (SNP) in the γ‐secretase component APH1B (Phe217Leu; rs1047552) was therefore analyzed for association with HIV‐1 infection. The SNP showed a tendency for association with HIV‐1 infection in a Xhosa indigenous South African Bantu study (P = 0.087), and associated significantly in a Caucasian Dutch study (P = 0.049). Together, the results suggest a role for the γ‐secretase pathway in susceptibility to HIV‐1 infection. J. Med. Virol. 81:1847–1851, 2009.