In the central nervous system (CNS), the oxidative deamination of monoamine neurotransmitters is accomplished by two membrane-bound enzymes: monoamine oxidase (of which there are two isoforms, MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO). The combined activities of these proteins are crucial for the regulation of neurotransmitter disposition and, consequently, normal brain function. It is therefore not surprising that MAO-A and B gene polymorphisms and altered expression are implicated in a variety of neurological disorders.1, 2, 3, 4, 5 Moreover, the demonstration that MAO inhibitors, such as iproniazid, were effective antidepressant agents was pivotal in Schildkraut's6 formulation of the catecholamine hypothesis of affective disorders. Here, we report for the first time the identification of a novel flavin adenine dinucleotide (FAD)-dependent protein, renalase, in various regions of the CNS. We show that the renalase gene is expressed in the hypothalamus and peripheral nerves. Furthermore, we reveal the existence of several splice variants of the renalase gene, which potentially serve to further regulate levels of monoamine neurotransmitters in the brain. Together, our findings provide further insight into the pathways regulating monoamine neurotransmitter disposition in the brain.
Until recently, it was thought that MAO and SSAO were the only monoamine oxidases expressed in humans. The discovery of a novel FAD-dependent protein, renalase, was reported in 2005.7 Renalase was identified using an in silico approach that aimed to discover novel proteins secreted by the kidney. The renalase protein sequence contains a highly conserved N-terminal FAD-binding domain and an amine oxidoreductase domain. Renalase shares low sequence identity with MAO-A and MAO-B (17 and 20 %, respectively) but, nonetheless, its predicted secondary and tertiary structures closely resemble those of MAO-B.8 Recombinant renalase was shown to generate hydrogen peroxide in the presence of monoamines (including catecholamines), suggesting that it may share the catecholamine-degrading activity of MAO-A and B.7 This activity was greatest in the presence of dopamine (followed by adrenaline and noradrenaline),7 but it was not inhibited by MAO inhibitors, indicating differences in the possible catecholamine-degrading actions of these proteins. The renalase sequence does not contain a membrane-tethering domain, indicating that, unlike MAO-A, B and SSAO, it may be responsible for the oxidation of monoamines in the cytosol or extracellular space. Renalase was identified primarily in the human kidney and heart, and was shown to be present in the plasma of healthy individuals.7 Although it has been suggested that renalase could not contribute to catecholamine degradation in plasma,9 renalase clearly has an important role in mammalian physiology, as shown by its ability to significantly improve hemodynamic parameters in animal models.7
Given the putative functional similarities between renalase and MAO-A and B, we investigated whether the tissue expression pattern of these proteins overlapped by searching for evidence of renalase expression in tissues that have earlier been shown to express MAO-A and B. Human tissue samples were obtained from the Victorian Institute of Forensic Medicine Tissue Donor Bank at autopsy. The informed consent of the donor's next of kin was obtained before the autopsy. All protocols were approved by the Victorian Institute of Forensic Medicine Ethics Review Committee. The length of time between death and autopsy did not exceed 72 h. Tissues were obtained from donors, male and female, who died from a variety of causes, including suicide, motor vehicle accident and drug overdose. Tissues were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The tissue powder was resuspended in radioimmunoprecipitation assay tissue lysis buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitors (protease inhibitor cocktail, Roche, Basel, Switzerland), homogenized using a 25-G needle and then incubated on ice for 1 h. Soluble protein was obtained by centrifugation at 10 000 × g for 10 min at 4 °C. Western blot analysis of soluble proteins was performed using a monoclonal antibody (from Ren Pharmaceuticals, CA, USA) raised against a 21 amino acid segment of the renalase protein. Results from various human tissues showed that, in addition to the heart and kidney, renalase is present in the forearm vein and artery, renal vein and artery and the ureter (Figure 1Ai). We also observed a signal for renalase in the hypothalamus and median nerve (see Figure 1Aii). A more thorough analysis of the distribution of renalase in the CNS revealed that, in addition to the hypothalamus, renalase is found in the pons, medulla oblongata, cerebellum, pituitary gland, cortex and spinal cord (Figure 1Aiii).
Having identified evidence of renalase protein synthesis in various tissues, reverse transcriptase PCR was used to confirm renalase gene expression in the hypothalamus and other tissues. Total RNA was extracted from tissue using the RNEasy system from Qiagen (Germantown, MD, USA). cDNA was generated using the Superscript III cDNA synthesis kit according to the manufacturer's instructions using 1 μg of total RNA (Invitrogen, Carlsbad, CA, USA). PCR was performed using renalase-specific primers (forward: 5′-ATGGCGCAGGTGCTGATCGTGGGC-3′; reverse: 5′-CTAAATATAATTCTTTAAAGCTTCCAG-3′) under the following conditions: 95 °C for 15 min, 35 cycles of 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Left ventricle RNA was used as a positive control. In the tissues examined (hypothalamus, adrenal gland and left ventricle), the major PCR product was of the expected size of 1026 bp. Three smaller PCR products were observed, sequenced and identified as splice variants of the renalase gene. Variant 1 (851 bp) did not contain exon 6. Instead, an alternative splicing between exon 5 and exon 7 resulted in a frameshift in the coding sequence of exon 7 and a premature stop codon. Variant 2 (777 bp) did not contain exons 2 and 3. In variant 3 (549 bp), exons 2, 3 and 6 were absent. We identified variants 2 and 3 in all tissue types examined (Figure 1Bi). Only the hypothalamus appeared to express little to no variant 1, suggesting that there is tissue-specific regulation of renalase function. Furthermore, an examination of hypothalamic transcripts revealed that there are differences in the relative amounts of wild type and variant 2 transcripts between donors (Figure 1Bii); however, these differences could not be ascribed to gender or mode of death. Whether this relates to a difference in renalase function in these individuals and whether the activity of the splice variants differs from that of wild-type renalase warrants further investigation.
Our identification of renalase, a soluble monoamine oxidase in the brain, highlights the possible existence of other pathways regulating monoamine neurotransmitter levels in the CNS. Inhibition of renalase activity may represent a novel target in the future design of therapeutics in the treatment of psychiatric disorders.
Conflict of interest
The authors declare no conflict of interest.
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Hennebry, S., Eikelis, N., Socratous, F. et al. Renalase, a novel soluble FAD-dependent protein, is synthesized in the brain and peripheral nerves. Mol Psychiatry 15, 234–236 (2010). https://doi.org/10.1038/mp.2009.74
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