BBOX1 is down-regulated in maternal immune-activated mice and implicated in genetic susceptibility to human schizophrenia
Introduction
Maternal immune activation (MIA) during pregnancy has been implicated in the etiologies of developmental neuropsychiatric disorders, including bipolar disorder (Canetta et al., 2014a, Canetta et al., 2014b), autism (Brown et al., 2014), and schizophrenia (Fatemi et al., 2008, Canetta et al., 2014a, Canetta et al., 2014b). MIA during pregnancy can be induced by viral mimic polyriboinosinic–polyribocytidilic acid (poly I:C) through injection, leading to a wide spectrum of schizophrenia-relevant functional and neuropathological deficits in adult offspring (Meyer et al., 2010a, Meyer et al., 2010b).
Gamma-butyrobetaine hydroxylase (BBOX 1), also known as gamma-BBH, BBH, G-BBH, and BBOX, is an enzyme encoded by the gene BBOX 1 in humans. Among rat, mouse, and human BBOX 1 nucleotide sequences, more than 80% are shared (Caroline et al., 2006). BBOX 1 belongs to the class of enzymes in the family of dioxygenases (Hausinger, 2004), also known as 2-oxoglutarate-dependent dioxygenases. They can catalyze the conversion from 2-oxoglutarate to succinate and CO2 concomitantly with oxygenating other substrates (Lindstedt et al., 1967, Lindstedt et al., 1968). These enzymes generally require Fe2+, a reducing cofactor, and ascorbic acid (Rebouche, 1991). BBOX 1 is the last enzyme involved in carnitine biosynthesis (Stéphane et al., 1998), which involves five enzymatic reactions that take two amino acids of ultimate precursors such as lysine and methionine (Vaz and Wanders, 2002). Lysine provides the carbon backbone of carnitine (Robert et al., 2010), while methionine provides the methyl group (Tanphaichitr et al., 1971). In the final step of enzyme reaction, gamma-butyrobetaine (GBB) is hydroxylated by BBOX to carnitine at three positions. BBOX is a cytosolic enzyme (Fre´de´ric et al., 1998) due to its main localization in the cytosol (Caroline et al., 2006, Vaz and Wanders, 2002, Tanphaichitr et al., 1971, Labadie et al., 1976). In these steps, carnitine is required for transporting long-chain fatty acids. Acetyl-L-carnitine is supplemented from L-carnitine (Traina et al., 2008) to the mitochondrial matrix for subsequent cellular energy production during regular metabolic activity (Judith et al., 2010). Short- and medium-chain acetyl esters formed in the mitochondria and peroxisome are involved in removing organic acids (Rebouche and Seim, 1998), and these reaction locations are the main positions responsible for BBOX activity in rats (Erfle, 1975, Paul et al., 1992); these reaction locations have also shown some BBOX activity in the human brain. The highest activity has been found in the kidney. BBOX activity is also present in the liver but not in the heart or muscle (Rebouche and Engel, 1980). In the brain, the role of carnitine in isotonicity is crucial because changing tonicity affects nerve excitability due to ion fluctuation (Peluso et al., 2000). One suggested mechanism of carnitine action is that it can reduce serum ammonia levels, thus improving psychometric measures (Malaguarnera et al., 2003, Malaguarnera et al., 2006, Malaguarnera et al., 2005). Acetyl-L-carnitine is neuroprotective when it is administered at supraphysiological concentrations (Traina et al., 2008), and it has clinical application in various neural disorders such as brain diseases and painful neuropathies (Traina et al., 2008). An increasing number of studies have demonstrated that secondary antioxidants such as acetylcarnitine can reduce or block neuronal death involved in the pathophysiology of brain disease. It has been suggested that mechanisms beyond antioxidant activities play a neuroprotective role (Mancuso et al., 2007).
Despite the potentially important neurobiological roles of BBOX 1, the association between BBOX 1 protein and pathophysiology of schizophrenia has not been reported yet. Therefore, the objective of this study was to investigate whether BBOX 1 protein expression was related to an animal model of schizophrenia and whether genetic polymorphism of BBOX 1 was associated with schizophrenia in a Korean population. For the study, we developed a mouse model of schizophrenia induced by repeated injection of polyI:C to examine the BBOX 1 expression levels in specific brain regions such as the medial prefrontal cortex (mPFC). We also performed pre-pulse inhibition test (PPI) for operational measure of sensorimotor gating. Furthermore, we screened for genetic variations near the 5′ region of BBOX 1 by direct sequencing and selected three single-nucleotide polymorphisms (rs7939644, rs1102795, rs10767592). Although these three SNPs have been reported in BBOX 1 (http://www.ensembl.org), they have not yet been investigated in the context of genetic association with schizophrenia. To investigate the association between near the 5′ region SNPs of BBOX 1 and schizophrenia in a Korean population, we conducted genotype, allele, and haplotype analyses to assess the associations between these SNPs and schizophrenia. We then performed a luciferase activity assay to determine the functional effects of these identified SNPs.
Section snippets
Animal model of schizophrenia
C57BL6/J mice (8 weeks old) were purchased (N = 10, males/N = 20, females) from Central Lab Animal Inc. (Seoul, Korea). Mice were mated in groups of one male and two females. When we observed a vaginal plug during daily control, the female mice were considered pregnant and separated. Pregnant dams on gestation day (GD) 9 received either a single injection of poly I:C or CON (saline) solution intravenously at the tail vein under mild physical constraint. Poly I:C (potassium salt) was obtained
Results
We detected significant pre-pulse facilitation in poly I:C-treated mice at lower pre-pulse stimulus intensities, but we detected no change in startle response in the non-polyI:C group (Fig. 1). We measured PPI using the acoustic startle response test in the offspring of the non-polyI:C and polyI:C treatment groups, and the results showed significant (P < 0.05) differences in PPI between the groups, specifically, mean pre-pulse stimulus levels of 3, 6, and 12 dB above background. However, PPI was
Discussion
To the best of our knowledge, this study is the first one that investigates the potential effect of BBOX 1 protein and human gene polymorphisms on schizophrenia. Our results showed that the poly I:C-induced schizophrenia animal model had decreased sensorimotor gating function with downregulated BBOX 1 protein. The results of genetic association study suggested that BBOX 1 polymorphisms might have increased schizophrenia susceptibility in the Korean population. The rs7939644 AA genotype in
Acknowledgements
The present study was performed with the support of Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03931619).
Conflicts of interest
The authors declare that they have no competing interests.
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