Family-based association analyses of imputed genotypes reveal genome-wide significant association of Alzheimer's disease with OSBPL6, PTPRG, and PDCL3.

The genetic basis of Alzheimer's disease (AD) is complex and heterogeneous. Over 200 highly penetrant pathogenic variants in the genes APP, PSEN1, and PSEN2 cause a subset of early-onset familial AD. On the other hand, susceptibility to late-onset forms of AD (LOAD) is indisputably associated to the ɛ4 allele in the gene APOE, and more recently to variants in more than two-dozen additional genes identified in the large-scale genome-wide association studies (GWAS) and meta-analyses reports. Taken together however, although the heritability in AD is estimated to be as high as 80%, a large proportion of the underlying genetic factors still remain to be elucidated. In this study, we performed a systematic family-based genome-wide association and meta-analysis on close to 15 million imputed variants from three large collections of AD families (~3500 subjects from 1070 families). Using a multivariate phenotype combining affection status and onset age, meta-analysis of the association results revealed three single nucleotide polymorphisms (SNPs) that achieved genome-wide significance for association with AD risk: rs7609954 in the gene PTPRG (P-value=3.98 × 10-8), rs1347297 in the gene OSBPL6 (P-value=4.53 × 10-8), and rs1513625 near PDCL3 (P-value=4.28 × 10-8). In addition, rs72953347 in OSBPL6 (P-value=6.36 × 10-7) and two SNPs in the gene CDKAL1 showed marginally significant association with LOAD (rs10456232, P-value=4.76 × 10-7; rs62400067, P-value=3.54 × 10-7). In summary, family-based GWAS meta-analysis of imputed SNPs revealed novel genomic variants in (or near) PTPRG, OSBPL6, and PDCL3 that influence risk for AD with genome-wide significance.

No association between CTNNBL1 and episodic memory performance.

Polymorphisms in the gene encoding catenin-ß-like 1 (CTNNBL1) were recently reported to be associated with verbal episodic memory performance--in particular, delayed verbal free recall assessed between 5 and 30 min after encoding--in a genome-wide association study on healthy young adults. To further examine the genetic effects of CTNNBL1, we tested for association between 455 single-nucleotide polymorphisms (SNPs) in or near CTNNBL1 and 14 measures of episodic memory performance from three different tasks in 1743 individuals. Probands were part of a population-based study of mentally healthy adult men and women, who were between 20 and 70 years old and were recruited as participants for the Berlin Aging Study II. Associations were assessed using linear regression analysis. Despite having sufficient power to detect the previously reported effect sizes, we found no evidence for statistically significant associations between the tested CTNNBL1 SNPs and any of the 14 measures of episodic memory. The previously reported effects of genetic polymorphisms in CTNNBL1 on episodic memory performance do not generalize to the broad range of tasks assessed in our cohort. If not altogether spurious, the effects may be limited to a very narrow phenotypic domain (that is, verbal delayed free recall between 5 and 30 min). More studies are needed to further clarify the role of CTNNBL1 in human memory.

Role of common and rare APP DNA sequence variants in Alzheimer disease.

OBJECTIVES: More than 30 different rare mutations, including copy number variants (CNVs), in the amyloid precursor protein gene (APP) cause early-onset familial Alzheimer disease (EOFAD), whereas the contribution of common APP variants to disease risk remains controversial. In this study we systematically assessed the role of both rare and common APP DNA variants in Alzheimer disease (AD) families. METHODS: Families with EOFAD genetically linked to the APP region were screened for missense mutations and locus duplications of APP. Further, using genome-wide DNA microarray data, we examined the APP locus for CNVs in a total of 797 additional early- and late-onset AD pedigrees. Finally, 423 single nucleotide polymorphisms (SNPs) in the APP locus, including 2 promoter polymorphisms previously associated with AD risk, were tested in up to 4,200 individuals from multiplex AD families. RESULTS: Analyses of 8 21q21-linked families revealed one family carrying a nonsynonymous mutation in exon 17 (Val717Leu) and another family with a partially penetrant 3.5-Mb locus duplication encompassing APP. CNV analysis in the APP locus revealed an additional family carrying a fully penetrant 380-kb duplication, merely spanning APP. Last, contrary to previous reports, association analyses of more than 400 different SNPs in or near APP failed to show significant effects on AD risk. CONCLUSION: Our study shows that APP mutations and locus duplications are a very rare cause of EOFAD and that the contribution of common APP variants to AD susceptibility is insignificant. Furthermore, duplications of APP may not be fully penetrant, possibly indicating the existence of hitherto unknown protective genetic factors.

Pharmacological activity and safety profile of P10358, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease.

1-[(3-Fluoro-4-pyridinyl)amino]-3-methyl-1(H)-indol-5-yl methyl carbamate (P10358) is a potent, reversible acetylcholinesterase inhibitor that produces central cholinergic stimulation after oral and parental administration in rats and mice. P10358 is a 2.5 times more potent acetylcholinesterase inhibitor than THA in vitro (IC50 = 0.10 +/- 0.02 microM vs. IC50 = 0.25 +/- 0.03 microM). It also inhibits butyrylcholinesterase activity as potently as THA (IC50 = 0.08 +/- 0.05 microM vs. IC50 = 0.07 +/- 0.01 microM). Ex vivo, P10358 (0.2 - 20 mg/kg, p.o.) produced dose-dependent inhibition of brain acetylcholinesterase activity. At 10 and 20 mg/ kg, it produced profound and long-lasting hypothermia in mice. P10358 enhanced performance in rats in a step-down passive avoidance task (0.62 and 1.25 mg/kg) and in a social recognition paradigm (0.32, 0.64 and 1.25 mg/kg) in mice. It reversed scopolamine-induced deficits in the Morris Water maze in rats (1.25 and 2.5 mg/kg) and a higher dose elevated striatal homovanillic acid levels. These behavioral and biochemical effects are consistent with central cholinergic stimulation. Hemodynamic studies in the rat demonstrated a 16-fold separation between behaviorally active doses (1.25 mg/kg) and those that elevated arterial pressure (20 mg/kg). Lethality in rats occurred at an oral dose of 80 mg/kg, but not at lower doses. Chemically, P10358 is an N-aminoindole and may not have the hepatotoxic liability associated with aminoacridine structure of tacrine. P10358 had weak affinity (>10 microM) at a variety of aminergic and peptidergic receptors and uptake carriers. These properties suggest that P10358 may be a safe and promising symptomatic treatment for Alzheimer's disease.

Iloperidone binding to human and rat dopamine and 5-HT receptors.

Iloperidone (HP 873; 1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy] -3- methoxyphenyl]ethanone) is a compound currently in clinical trials for the treatment of schizophrenia. Iloperidone displays affinity for dopamine D2 receptors and for 5-HT2A receptors and has a variety of in vivo activities suggestive of an atypical antipsychotic. Here we present an examination of the affinity of iloperidone to a variety of human and rat homologs of dopamine and 5-HT receptor subtypes. We employed receptor binding assays using membranes from cells stably expressing human dopamine D1, D2S, D2L, D3, D4 and D5 and 5-HT2A and 5-HT2C receptors and rat 5-HT6 and 5-HT7 receptors. Iloperidone displayed higher affinity for the dopamine D3 receptor (Ki = 7.1 nM) than for the dopamine D4 receptor (Ki = 25 nM). Iloperidone displayed high affinity for the 5-HT6 and 5-HT7 receptors (Ki = 42.7 and 21.6 nM, respectively), and was found to have higher affinity for the 5-HT2A (Ki = 5.6 nM) than for the 5-HT2C receptor (Ki = 42.8 nM). The potential implications of this receptor binding profile are discussed in comparison with data for other antipsychotic compounds.

Structure-activity relationships of a series of novel (piperazinylbutyl)thiazolidinone antipsychotic agents related to 3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2,5,5- trimethyl-4-thiazolidinone maleate.

HP-236 (3-[4-[4-(6-Fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2,5,5- trimethyl-4-thiazolidinone maleate; P-9236) (54) displayed a pharmacological profile indicative of potential atypical antipsychotic activity. A series of piperazinyl butyl thiazolidinones structurally related to this compound were prepared and evaluated in vitro for dopamine D2 and serotonin 5HT2 and 5HT1A receptor affinity. The compounds were examined in vivo in animal models of potential antipsychotic activity and screened in models predictive of extrapyramidal side effect (EPS) liability. The synthesis of these compounds, details of their structure-activity relationships, and discovery of a new lead, compound 50, as well as further development of the profiles of compounds 50 and 54 are described.

Ex vivo studies with iloperidone (HP 873), a potential atypical antipsychotic with dopamine D2/5-hydroxytryptamine2 receptor antagonist activity.

Iloperidone {HP 873: 1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy] -3-methoxyphenyl]ethanone} is a dopamine (D2)/serotonin (5-HT2) receptor antagonist with the preclinical profile of an atypical antipsychotic based on biochemical studies in rats. Iloperidone significantly increased dopa accumulation, an index of dopamine turnover in response to D2 receptor blockade, at doses from 0.3 to 10 mg/kg i.p. in the striatum and from 1 to 10 mg/kg in the nucleus accumbens. Blockade of dopaminergic presynaptic autoreceptors was measured by the reversal of apomorphine-inhibition of gamma-butyrolactone-induced dopa synthesis. Iloperidone did not significantly reverse the apomorphine inhibition of gamma-butyrolactone-induced dopa synthesis at any of the doses tested (0.3-10 mg/kg i.p.). In ex vivo receptor autoradiography studies, a 30-min pretreatment with iloperidone (2.5-20 mg/kg i.p.) inhibited the binding of [3H]spiperone to cortical and subcortical 5-HT2 receptors by 42 to 94%, in contrast to only 1 to 15% inhibition of [3H]spiperone binding to D2 receptors in the nucleus accumbens and striatum. Iloperidone, at 2.5 mg/kg i.p., inhibited 5-HT2 receptor binding by 54 to 62% at 4-hr post-treatment, whereas there was negligible inhibition of D2 receptors. Chronic treatment with 5 mg/kg i.p. of iloperidone for 19 days significantly decreased the number of 5-HT2 receptors in the frontal cortex with no change in receptor affinity. D2 receptor number and affinity were unchanged in the nucleus accumbens and six regions of the striatum. In summary, iloperidone is a 5-HT and dopamine receptor antagonist with weak activity at presynaptic dopamine autoreceptors. Potent 5-HT2 receptor antagonism may be an important component in the preclinical profile of iloperidone as a potential atypical antipsychotic.

Serotonergic activity of HP 184: does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl) -1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 microM HP 184 enhanced spontaneous release of [3H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30-40% after a 14-21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain:plasma ratio of HP 184 was approximately 2:1, with brain concentrations of 1.6 micrograms/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.

Besipirdine (HP 749) reduces schedule-induced polydipsia in rats.

The aim of the present paper is to report on the adrenergic and serotonergic effects of besipirdine (HP 749) in vivo and to discuss its potential use in the treatment of obsessive compulsive disorder. Besipirdine inhibited biogenic amine uptake in vitro. It prevented tetrabenazine-induced ptosis in mice and potentiated the 5-hydroxytryptophan-induced serotonin syndrome in rats. Furthermore, it decreased schedule-induced polydipsic behavior in rats. Schedule-induced polydipsia may be a model for obsessive compulsive disorder. Previous results from our group have shown that certain selective serotonin reuptake inhibitors decrease schedule-induced polydipsia after 14-21 days of treatment. Besipirdine reduced schedule-induced polydipsic behavior immediately and this reduction lasted throughout the duration of the experiment (29 days).

Changes in paroxetine binding in the cerebral cortex of polydipsic rats.

Schedule-induced polydipsia was induced when food-deprived rats were subjected to a fixed-time (60 s) feeding schedule for 150 min daily for 3 weeks (training period). Subsequent chronic administration of the serotonin reuptake inhibitor fluoxetine reduces schedule-induced polydipsia over 2-4 weeks. We asked whether changes in the serotonin reuptake carrier occur following the development of schedule-induced polydipsia and its reduction by fluoxetine. Using [3H]paroxetine binding, we found a 40% increase in Kd and a 50% decrease in Bmax in polydipsic rats; both were reversed by fluoxetine. Food deprivation alone did not affect these parameters. These observations suggest that changes in the serotonin reuptake carrier correlate with the development and reversal of schedule-induced polydipsia.

Benzisoxazole- and benzisothiazole-3-carboxamides as potential atypical antipsychotic agents.

A series of benzisoxazole- and benzisothiazole-3-carboxamides has been prepared and tested for potential antipsychotic activity. In general, the compounds showed an affinity for dopamine D2 and serotonin 5HT2A and 5HT1A receptors. Several members of this series have demonstrated activity in animal models predictive of potential antipsychotic activity. In addition, compounds 18, 19, 22, 27, 28, 43, and 44 have also shown a potential for reduced EPS liability as suggested by the ratio of activity seen in mesolimbic-mediated vs nigrostriatal-mediated behavioral assays.