Investigation of endocannabinoid system genes suggests association between peroxisome proliferator activator receptor-α gene (PPARA) and schizophrenia

https://doi.org/10.1016/j.euroneuro.2012.07.007Get rights and content

Abstract

Schizophrenia (SZ) is a complex psychiatric disorder with a large genetic burden and an estimated hereditability of 80%. A large number of neuroanatomical and psychopharmacological studies suggest a central role of the endocannabinoid (eCB) system in the susceptibility of the disease. To further investigate this hypothesis, we performed an association study with genes codifying for key elements of the eCB system in a sample of 170 schizophrenic patients and 350 healthy controls of Italian ancestry. A total of 57 Tag SNPs (tSNPs) were selected using HapMap CEU population SNP database spanning the following genes: cannabinoid receptor 1 (CNR1), peroxisome proliferator activator receptor-α (PPARA), fatty acid amide hydrolase (FAAH) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). Seven out of the 32 tSNPs within PPARA (rs4253765, rs4263776, rs6007662, rs1800206, rs4253763, rs6008197 and rs4253655) and 3 out of 12 tSNPs within CNR1 (rs1049353, rs7766029 and rs806366) were nominally associated with SZ (uncorrected p<0.05). The same pattern of association was observed in the genotype analysis, with rs4253765 showing the highest level of significance (uncorrected p=2×10−3). None of these associations survived after permutation test. Our findings suggest a potential role for PPARA in the susceptibility to SZ, but further studies on larger independent samples are warranted in order to clarify the involvement of this gene in the pathophysiology of SZ.

Introduction

Schizophrenia (SZ) is a severe and debilitating psychiatric disease often with a chronic course, affecting approximately 1% of the world population (Sadock et al., 2009). Although its etiology is still unclear, a large number of evidence has strongly suggested that biological alterations with an underlying genetic basis play a key role in SZ (Ross et al., 2006). So far, several etiological models have been proposed for SZ including dopaminergic, GABAergic and neurodevelopmental hypotheses. During the last decade, a growing body of findings has also suggested that the endogenous cannabinoid (eCB) system might be altered in SZ patients (Parolaro et al., 2010). Converging evidence have shown that Cannabis abuse, especially in vulnerable ages, is a risk factor for SZ, being associated with a higher incidence of the disease in adulthood (Arseneault et al., 2002, Caspi et al., 2005). Interestingly, Cannabis use is more frequent in patients suffering from SZ compared to healthy individuals (Kovasznay et al., 1997) and the abuse of Cannabis can precipitate the psychotic state, with hallucinations and delusions resembling SZ in vulnerable subjects (Leweke et al., 2007) as well as worsen positive symptoms of patients with SZ (Linszen et al., 1994).

Altered activity of the eCB system in SZ patients has been supported by a number of imaging studies.

Binding of the cannabinoid receptors-1 (CB1) selective PET radioligand [11C]OMAR is elevated across several brain regions of SZ subjects compared to controls, especially in the pons (Wong et al., 2010). A recent finding also showed an increase in CB1 availability in the mesocorticolimbic circuitry, particularly in the nucleus accumbens of SZ patients, independent of the treatment with antipsychotic (Ceccarini et al., 2010). This study also showed that SZ patients treated with antipsychotic monotherapy present increased relative CB1 binding in the insula and anterior cingulate cortex (Ceccarini et al., 2010).

The eCB dysregulation in SZ is also supported by postmortem studies, showing an increased density of CB1, particularly in the dorso-lateral prefrontal cortex of SZ subjects compared to controls, independent of recent intake of Cannabis (Dean et al., 2001).

Clinical and laboratory findings have also suggested that variations in eCB levels may be implicated in SZ, reporting significantly higher levels of the N-acylethanolamines (NAEs), anandamide (AEA) and palmitoylethanolamide (PEA), in cerebrospinal fluid (Giuffrida et al., 2004, Leweke et al., 1999) and blood (De Marchi et al., 2003) of SZ patients compared to healthy volunteers. AEA is one of the best studied “classical” endogenous ligands for CB1 (Devane et al., 1992), whereas PEA, and its congener oleoylethanolamide (OEA), are structural analogs of AEA and share the main synthesizing and catabolic enzymes, the N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), respectively (Ueda et al., 2000). PEA and OEA constitute a parallel endocannabinoid-like system (Mackie and Stella, 2006) and belong to the family of endogenous agonists at peroxisome proliferator-activated receptors-alpha (PPAR-α) (Fu et al., 2003), but do not activate CB1 (Pistis and Melis, 2010).

PEA has long been known as an antinflammatory (Benvenuti et al., 1968) and neuroprotective lipid, whereas OEA (but also PEA with less potency) was demonstrated to reduce feeding and body weight gain (Fu et al., 2003) through PPAR-α activation.

Interestingly, both typical eCBs and eCB-like lipids are endogenous modulators of dopamine (DA) transmission via CB1 or PPAR-α, respectively. Hence, eCBs are released by ventral tegmental area (VTA) DA neurons (Melis et al., 2004) and regulate the strength of impinging GABA and glutamate synapses acting retrogradely at presynaptic CB1 (Melis and Pistis, 2007). Likewise, recent evidence suggests that OEA and PEA modulate DA neurons in autocrine-like fashion (Melis et al., 2010, Melis et al., 2008). In particular, they depress firing rate of DA cells in the VTA and the number of spontaneously active DA neurons through a rapid non-genomic mechanism downstream to activation of PPAR-α (Melis et al., 2010). The authors speculated that PPAR-α, a nuclear receptor which regulates the transcription of genes involved in fatty acid oxidation, extracellular lipid metabolism, homeostasis and inflammation (Fruchart et al., 1999) might be involved in disorders in which DA dysfunction plays a prominent role, such as SZ.

It is known that D2 receptors stimulation induces an increased release of AEA, counterbalancing dopamine-mediated psychotic symptoms (Giuffrida et al., 2004), but how OEA and PEA are synthesized by DA neurons, and their role in the neurobiology of SZ, are still under investigation. This functional interaction between AEA, OEA/PEA and dopamine integrates the cannabinoid hypothesis in the dopaminergic hypothesis of SZ. Moreover, it has been suggested that raised levels of AEA and PEA act as an acute reactive or “protective” mechanism during acute psychoses (Leweke et al., 2007).

Rapid termination of NAE signaling in the central nervous system is ensured by the metabolic activity of FAAH (Cravatt et al., 1996). Elevated mRNA plasmatic levels of FAAH were reported in SZ patients (De Marchi et al., 2003) while Malone et al. (2008), using the model of social isolation in rats, found a significant increase in FAAH protein levels in several brain areas involved in SZ. Moreover, an increase in mRNA expression levels of CB1 and NAPE-PLD was observed, particularly in the prefrontal regions, cortical layers and a number of thalamic regions of socially isolated rats (Robinson et al., 2010).

Despite the large body of evidence towards the involvement of eCB-related elements in SZ, genetic studies have thus far marginally investigated the role of eCB genes in the susceptibility to the disorder or related endophenotypes. The CB1 receptor gene (CNR1) has been the most studied, but results have been generally elusive (Chavarría-Siles et al., 2008, Hamdani et al., 2008, Ujike et al., 2002). However, it is of interest to note that the region containing CNR1, located on chromosomes 6q14-q15, has been suggested as a susceptibility locus for SZ in a linkage study (Cao et al., 1997).

The association of other genes of the eCB system with SZ has been investigated by a small number of studies and further investigation would be needed to understand their role in the susceptibility to the disease (Ishiguro et al., 2010, Morita et al., 2005).

Based on the aforementioned evidence, we carried out a case-control association study in a sample of SZ patients and controls of Italian origin by selecting 57 Tag Single Nucleotide Polymorphisms (tSNPs) located in PPARA, CNR1, FAAH and NAPE-PLD genes with the aim of further exploring and elucidating the role of the eCB system in SZ.

Section snippets

Study subjects

Our sample comprised 170 schizophrenic patients [119 males and 51 females, mean age (years±SD) 34±7.6] and 350 healthy controls [246 males and 104 females, mean age (years±SD) 36±9.6]. Patients were diagnosed according to DSM-IV criteria and the Structured Clinical Interview for DSM-IV (SCID-I) and were recruited at the outpatient units, day-care programs and rehabilitation centers of the University Psychiatric Departments of Naples, Milan, L'Aquila, Pisa and the Section of Clinical

Demographic features and quality control

Cases and controls did not differ in terms of age or gender distribution (p<0.05). However, the ratio of males to females was higher both in cases and controls, being 2.3 and 2.4 respectively. We therefore tested whether gender was a confounding factor, reporting no significant effect on association values (data not shown). No significant deviation from the HWE was observed in cases and controls (HWE>0.001).

SNPs rs1477160 in NAPE-PLD and rs761543, rs6008976, rs4253682, rs4253790, rs4253783 in

Discussion

Based on the evidence indicating the possible involvement of eCB and eCB-like systems in SZ, we meant to investigate the role of genetic variants within PPARA, CNR1, FAAH and NAPE-PLD in the predisposition to the disorder.

We reported significant associations for 7 of the 28 SNPs analyzed in PPARA, with rs4253765 showing the strongest association. While the significance did not stand the permutation testing, it suggests a potential involvement of PPARA in the pathophysiology of SZ.

Although a

Role of funding source

This research was partly supported by a Grant from the Regional Councillorship of Health (Regione Autonoma della Sardegna L.R. n. 7/2007 Bando 2009) for the Promotion of Scientific Research and Technological Innovation in Sardinia, and by Grant no. 9806499118 from the Italian Ministry of University and Scientific Research, 2003.

Funding agencies had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit

Contributors

M.C. and D.C. realized the experiments, performed the statistical analysis and wrote the first draft of the manuscript.

A.S. contributed to the experimental design, supervised the first draft of the manuscript and revised the manuscript according to referee's comments.

P.N. carried out a part of the experiments.

C.C. realized and analyzed the clinical database.

S.G. coordinated the clinical work.

M.P. supervised the manuscript as an expert neuroscientist.

M.D.Z. designed the study, coordinated the

Conflict of interest

All authors declare that they have no conflicts of interest.

Acknowledgments

The authors wish to thank Nurse Flavia Orrù for her collaboration in the clinical work.

References (53)

  • M. Mameli-Engvall et al.

    Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors

    Neuron

    (2006)
  • M. Melis et al.

    Peroxisome proliferator-activated receptors-alpha modulate dopamine cell activity through nicotinic receptors

    Biol. Psychiatry

    (2010)
  • S. Moreno et al.

    Immunolocalization of peroxisome proliferator-activated receptors and retinoid X receptors in the adult rat CNS

    Neuroscience

    (2004)
  • Y. Morita et al.

    A nonsynonymous polymorphism in the human fatty acid amide hydrolase gene did not associate with either methamphetamine dependence or schizophrenia

    Neurosci. Lett.

    (2005)
  • D. Parolaro et al.

    The endocannabinoid system and psychiatric disorders

    Exp. Neurol.

    (2010)
  • S.A. Robinson et al.

    The effect of social isolation on rat brain expression of genes associated with endocannabinoid signaling

    Brain Res.

    (2010)
  • C.A. Ross et al.

    Neurobiology of schizophrenia

    Neuron

    (2006)
  • A. Schmitt et al.

    Altered thalamic membrane phospholipids in schizophrenia: a postmortem study

    Biol. Psychiatry

    (2004)
  • J. Seifert et al.

    No association of CNR1 gene variations with susceptibility to schizophrenia

    Neurosci. Lett.

    (2007)
  • N. Ueda et al.

    The fatty acid amide hydrolase (FAAH)

    Chem. Phys. Lipids

    (2000)
  • D.F. Wong et al.

    Quantification of cerebral cannabinoid receptors subtype 1 (CB1) in healthy subjects and schizophrenia by the novel PET radioligand [11C]OMAR

    Neuroimage

    (2010)
  • L. Arseneault et al.

    Cannabis use in adolescence and risk for adult psychosis: longitudinal prospective study

    BMJ

    (2002)
  • F. Benvenuti et al.

    Activity of some derivatives of palmitoylethanolamide on carragenine-induced edema in the rat paw

    Boll. Soc. Ital. Biol. Sper.

    (1968)
  • I. Chavarría-Siles et al.

    Cannabinoid receptor 1 gene (CNR1) and susceptibility to a quantitative phenotype for hebephrenic schizophrenia

    Am. J. Med. Genet. B Neuropsychiatr. Genet.

    (2008)
  • B.F. Cravatt et al.

    Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides

    Nature

    (1996)
  • V.S. Dalton et al.

    Paranoid schizophrenia is characterized by increased CB1 receptor binding in the dorsolateral prefrontal cortex

    Neuropsychopharmacology

    (2011)
  • Cited by (25)

    • Targeted lipidomics and metabolomics evaluations of cortical neuronal stress in schizophrenia

      2019, Schizophrenia Research
      Citation Excerpt :

      We therefore undertook a validation of these observations in a third cohort of post-mortem frontal cortex tissues and expanded our study to investigate N-acylphosphatidylethanolamines (NAPEs), which are well established biomarkers of neuronal stress in models of hypoxia/ischemia (Hansen et al., 2000; Kilaru et al., 2011; Janfelt et al., 2012; Luptakova et al., 2018) and models of excitotoxicity (Hansen et al., 1997; Hansen et al., 2001; Guan et al., 2006). The potential value of such analyses is further supported by reports of polymorphisms in NAPE-phospholipase D (NAPE-PLD; EC 3.1.4.54) and fatty acid amide hydrolase (FAAH; EC 3.5.1.99) in schizophrenia (Costa et al., 2013; Si et al., 2018). Alterations in NAPE-PLD, which cleaves NAEs (endocannabinoids) from NAPEs at sn-3, and in FAAH which regulates the levels of NAEs, are likely to contribute to decreased levels of NAEs in the cortex in schizophrenia (Muguruza et al., 2013).

    • Association of CNR1 genotypes with changes in neurocognitive performance after eighteen-month treatment in patients with first-episode psychosis

      2019, European Psychiatry
      Citation Excerpt :

      Interestingly, Wirtz et al. [29], reported an association between rs1049353 polymorphism and activation of prefrontal cortex when viewing negative pictures after stress (more in AA/AG then the GG genotype) and showed that memory performance correlated with amygdala and hippocampus activity and connectivity in stressed carriers of AA/AG but not the GG genotype, all suggesting more vulnerability/stronger affective response to negative stressors in the carriers of the A allele. On the other hand, the G allele was suggested as a risk variant in schizophrenia and cannabis abuse [14,53]. Following the same logic in our results, one possible explanation may be that the carriers of risk alleles, the CC rs7766029 and AG rs12720071 genotype, perceive their emotions less intensely, perhaps also due to negative symptoms of illness, and thus rated their perceived level of stress as lower compared with carriers of other variants, but are in fact more susceptible to FEP with a different genetic background (for example, non-affective psychosis vs. affective psychosis).

    • The endocannabinoid system in mental disorders: Evidence from human brain studies

      2018, Biochemical Pharmacology
      Citation Excerpt :

      Some other studies have analyzed the SNP rs1049353 in schizophrenic patients [87,146], none of them showing any significant linkage. Several other studies have failed in trying to find associations between different SNPs of CNR1 (rs806366, rs806368, rs806376, rs806379, rs806380, rs6454674, sr1535255 among other) and schizophrenia [147–150]. However, some SNPs such as rs6454674 [151], rs2023239 [152] and interactions with rs1049353, rs1535255, and rs2023239 [152] have been associated with positive and negative symptoms.

    • Brain structural and clinical changes after first episode psychosis: Focus on cannabinoid receptor 1 polymorphisms

      2015, Psychiatry Research - Neuroimaging
      Citation Excerpt :

      Also CNR1 polymorphisms have been associated with psychosis-related disorders (Eggan et al., 2008, 2012; Brown et al., 2014). However, some studies have failed to reveal a significant association between schizophrenia and CNR1 genetic mutations through studies of the rs1049353, rs77660229, rs86366 and rs6454674 polymorphisms (Seifert et al., 2007; Costa et al., 2013), whereas others have suggested that differences in the gene are related to schizophrenia vulnerability, independent of substance abuse (Martinez-Gras et al., 2006). Moreover, the rs1049353 polymorphism has been associated with predisposition to the hebephrenic schizophrenia subtype (Ujike et al., 2002).

    View all citing articles on Scopus
    1

    Contributed equally to the manuscript.

    View full text