Effect of long-term treatment with escitalopram on spexin, nesfatin-1 and S mRNA expression in the rat brain

Artur Pałasz1, Aleksandra Suszka-Świtek1, Katarzyna Bogus1, Łukasz Filipczyk1, Ewa Rojczyk1, Rafał Skowronek2, Ryszard Wiaderkiewicz1, Marek Krzystanek3

1Department of Histology, 2Department of Forensic Medicine and Toxicology, 3Department and Clinic of Psychiatric Rehabilitation

School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland

Background Methods overview

Spexin (SPX) is an intriguing novel neuropeptide, a product of NPQ gene which was identified through bioinformatics search strategy. In the brain, many SPX immunopositive neural populations were described, while neuroglia was usually negative, the highest reaction was detected in the hypothalamic paraventricular and supraoptic nuclei. The hippocampal neurons showed moderate immunoreactivity as well as cerebellar Purkinje cells and brainstem neurons [1]. SPX seems to have multiple physiological functions in mammals. Studies using goldfish revealed the involvement of SPX in reproduction and food-intake regulation. Treatment of animals with SPX decreased the secretion of LH and suppressed . Brain injection of goldfish SPX inhibited both basal and NPY- or -dependent food intake. Intriguing recent results published by Walewski et al. (2015) showed that SPX may be a novel strongly anorexigenic factor involved in weight regulation, with potential for obesity therapy [2]. A ligand-receptor interaction study suggested that SPX may be a natural ligand for i.p. GALR2/3 receptors. Nesfatin-1, a newly discovered NUCB2-derived satiety neuropeptide plays an important role in hypothalamic pathways Escitalopram regulating food intake and energy homeostasis. Nesfatin-1 immunoreactive cells are located mainly in arcuate, paraventricular and supraoptic nuclei, where the is colocalized with POMC/CART, NPY, and . Nesfatin-1 molecule interacts with a G-protein coupled receptor and its cytophysiological effect depends on inhibitory hyperpolarization of NPY/AgRP neurons in ARC and signaling in PVN. Administration of nesfatin-1 significantly inhibits consumatory behaviour and decreases weight gain in experimental animals. The peptide is also RT-PCR expressed in several neurons of forebrain, hindbrain, brainstem and spinal cord. The recent findings suggest the evidence for nesfatin-1 involvement in other important brain functions such as reproduction, sleep, mood, cognition and anxiety- or stress-related responses [3]. Neuropeptide S (NPS), a potent central anxiolytic factor, is also involved in the regulation of sleep/wake states, motor activity, energy balance as well as plays a role in the pathomechanism of addiction [4]. Some findings suggest a possible role for the NPS system in mediating physiological effects of caffeine and nicotine. NPS also mediates memory IHC consolidation mechanisms both aversive and neutral. NPS is expressed in the rat brain mainly within the , principal trigeminal and lateral parabrachial nuclei. A number of NPS–positive parabrachial cells coexpress corticotropin releasing factor (CRF) Moreover, most of the pericoerulear NPS-expressing neurons are glutamatergic, while only a small, laterally located group of cells show acetylocholine expression.

Quantitative Real-Time PCR Purpose Experiments were carried out on adult rats to assess long-term (28 days) effect of the Present study has an aim to examine (on animal model) if and how long-term treatment with escitalopram influence the escitalopram at dose 10 mg/kg on SPX, NPS, NPSR and mRNA expression in the brain. expression of spexin, nesfatin-1 and neuropeptide S (NPS) mRNA in the rat encephalon. In this experiment we measured Escitalopram is a S-enantiomer of citalopram, well known highly selective serotonin reuptake inhibitor for the first time the mRNA levels of aforementioned and spexin protein expression in the various brain (SSRI) very effective in treatment of depression and anxiety disorders. It does not bind to serotonin, structures after antidepressant administration. (D1 and D2), muscarinic and histamine receptors, that minimize the range of its potential side effects. Total mRNA was extracted from all samples via the phenol-chloroform method and dissolved in 50 μl of RNAse-free water. mRNA were transcribed into cDNA during incubation in buffered solution of reverse transcriptase MMLV-RT with RNAsin, oligo-dT and mix of nucleotides at 42 0C for 60 min. using a DNA Thermal Cycler 480 (Perkin Elmer Inc., Waltham, MA). Initial mRNA solutions contained 5 µg of RNA per 100 µl. Quantitative Real-Time PCR reaction (qPCR) was performed by FastStart SYBR Green Master (Roche) in Light Cycler ® 96 (Roche) thermal cycler for 40 rounds. Glyceraldehyde phosphate dehydrogenase (GAPDH) or beta-2-microglobulin were chosen as standard internal reference genes. cDNA was amplified using the TaqMan Gene Expression Assay Spexin (Rn01749065_m1, Applied Biosystems) and TaqMan Gene Expression Master mix (4369016, Applied Biosystems). Primer sequences for other neuropeptides: NUCB2: Forward: 5′ -TTTGAACACCTGAACCACCA-3′, Reverse: 5′-TGCAAACTTGGCTTCTTCCT 3′ NPS; Forward: 5 - TTGGAGTTATCCGGTCCTCTCTT -3, Reverse: TTGGAGTTATCCGGTCCTCTCTT-3, NPSR; Forward: 5- TGCAAGGTGCAAAGATCCCA-3, Reverse: 5-AATCTGCATCTCATGCCTCTC-3. Kiss-1: Forward: 5’ - TGGCACCTGTGGTGAACCCTGAAC -3’, Reverse: 5’ – GCCACCTGCCTCCTGCCGTAGCGC-3’; GAPDH: Forward: 5’–GTGAACGGATTT A. C. GGCCGTATCG–3’, Reverse: 5’–ATCACGCCACA GC TTTCCAGAGG-3. GAPDH: Forward: 5’– GTGAACGGATTTGGCCGTATCG–3’, Reverse: 5’–ATCACGCCACAGCTTTCCAGAGG-3. Neuroanatomical distribution of nesfatin-1 (A.) and NPS neurons (C., blue areas) in the rat brain. A group of NPS-immunopositive perikarya in the rat locus coeruleus (B.). ARC, arcuate nucleus; DMH, dorsomedial hypothalamus; LHA, lateral hypothalamus; PVN, paraventricular nucleus; SON, supraoptic nucleus; PeF, perifornical area; ZI, zona incerta; BarN, Barrington nucleus; 5M, motor trigeminal nucleus, LC, locus coeruleus; LPB, lateral Immunohistochemistry parabrachial nucleus, PrDM, dorsomedial part of principal trigeminal nucleus; Pr5VL, ventrolateral part of principal trigeminal nucleus, 5M motor trigeminal nucleus; RtTg, reticular tegmental nucleus; Su5, supratrigeminal nucleus; 4V, fourth ventricle. Brain slices were dehydrated and embedded in paraffin. Finally, they were sectioned on the microtome (Leica Microsystems, Germany) in the coronal plane (−2.00 to−2.80 mm from bregma) at 7 μm thickness, according to Paxinos & Watson’s The Rat Brain in Stereotaxic Coordinates (2007). After Results rehydratation, antigen retrieval and blockage with 10% serum, brain sections were incubated overnight in 4 °C with the rabbit antibody against rat spexin (1:500, Phoenix Pharmaceuticals), Incubation with primary antibodies was followed by administration of biotynylated anti-rabbit secondary antibodies (1:200) for 30min, and then an avidin-biotin-horseradish peroxidase complex (Vectastain ABC kit, Vector Labs) for another 30 min. Finally, 3,3′-diaminobenzidine (DAB) was used (Vectastain ABC kit, Vector Laboratories) to complete the reaction and visualize cells expressing particular neuropeptides. Sections incubated with rabbit IgG instead of primary antibody were used as negative controls.

Quantitative PCR results of relative NUCB2, NPS, NPSR and kisspeptin mRNA expression levels in the rat hypothalamus, Quantitative PCR results of relative spexin mRNA expression levels in the rat hypothalamus, hippocampus, , striatum, cerebellum and hippocampus, amygdala, striatum, cerebellum and brainstem. Obtained results were normalized to GAPDH reference gene. brainstem (top left). Obtained results were normalized to beta-2-microglobulin reference gene. Mean number of spexin immunoreactive cells in the Data are presented as mean ± SEM. three examined rat brain structures. Data are presented as multiples/decimals of control (1) ± SEM (bottom left). Representative expression of spexin in the rat hypothalamus, hippocampus and striatum in controls and after long-term treatment with escitalopram. Scale bars: 50 m (right side).

Ki-67 Conclusions - - We have demonstrated for the first time, that the extended treatment with an SSRI drug modulate the expression of SPX, NPS, NPSR and kisspeptin mRNAs in the- rat brain. On the other hand, escitalopram seems to not affect the nesfatin-1 signaling in the examined brain structures. The results reported may suggests a so far unknown relationships between these novel regulatory factors, which has implications for food intake control, HPA axis modulation, anxiety/stress responses and probably other CNS functions. The fact that neuroleptics affected the level of NPS and NPSR mRNA expression may support the hypothesis that NPS plays a role in the anxiolytic- actions of these commonly used medications and possibly also in the pathophysiology of depression as well as other mental disorders. At present, when antidepressant-related changes in peptide expression are not yet clarified, comparing actions of escitalopram on the measured mRNA levels in the brain regions to its effects on other neuropeptides e.g. is unfortunately merely speculative. Although satisfactory explanation of SPX and NPS functions requires numerous further- studies, even at the present stage of knowledge these substance can be also considered potentially important regulatory factor in the brain. Undoubtedly, this intriguing initial data requires further basic pharmacological studies on the wide spectrum of antidepressants, but nonetheless, highlights the complex nature of potential interactions between serotonin signaling and brain peptidergic pathways which opens up an array of future potential clinical applications. References [1] Porzionato A., Rucinski M., Macchi V., Stecco C., Malendowicz L.K., De Caro R. 2010. Spexin expression in normal rat tissues. J Histochem Cytochem 58, 825-37. [2] Walewski J.L., Ge F., Lobdell H. 4th, Levin N., Schwartz G.J., Vasselli J.R., Pomp A.,Dakin G., Berk P.D. 2014. Spexin is a novel human peptide that reduces adipocyte uptake of long chain fatty acids and causes weight loss in rodents with diet-induced obesity. Obesity 22, 1643-52. [3] Pałasz A., Krzystanek M., Worthington J., Czajkowska B., Kostro K., Wiaderkiewicz R., Bajor G. 2012. Nesfatin-1, a unique regulatory neuropeptide of the brain. Neuropeptides 46, 105-12. [4] Pape H.C., Jungling K. Seidenbecher T., Lesting J. Reinscheid R.K. 2010. Neuropeptide S: a transmitter system in the brain regulating fear and anxiety. Neuropharmacology 58, 29-34.