NEUROPEPTIDES IN THE HUMAN POSTMORTEM BRAIN FOCUS ON GALANIN AND ITS RECEPTORS IN MAJOR DEPRESSION

Neuropeptides represent the most diverse family of messenger molecules in the nervous system, which are co-expressed with key neurotransmitters in several brain regions. They modulate synaptic neurotransmission and have, in some cases, also trophic functions. One such neuropeptide, and the focus of the thesis, is ‘galanin’. It is a classical neuropeptide that functions in multiple (patho-) physiological processes, and exerts its actions via three receptors (GalR1-3). A main aim of the thesis is to establish the distribution of, mainly, the galanin system in the human, postmortem brain, but neuropeptide S (NPS) has also been analyzed. We further explore expression of the galanin family genes in postmortem brains from patients suffering from major depressive disorder (MDD). In Paper I we studied the localization of galanin and its three receptor transcripts primarily in the locus coeruleus (LC) and dorsal raphe nucleus (DRN) of ‘normal’, human postmortem brains using RNA in situ hybridization (ISH) and quantitative PCR (qPCR). In the rat brain galanin is known to co-exist with noradrenaline (NA) in most locus LC neurons and with 5hydroxytryptamine (5-HT) in many DRN neurons, two regions that are considered to be important in mood related disorders. In the human brain, galanin and GalR3 mRNA were found in many NA-LC neurons, and GalR3 mRNA overlapped with tryptophan hydroxylase 2 (TPH2) transcript in at least in some regions of the DRN. However, galanin may not be expressed in 5-HT neurons. qPCR analysis confirmed the expression levels and pattern above, suggesting that distinct species differences exist with regard to galanin and galanin receptor expression in the human brain, when compared to the rat. Such differences are important when defining targets for drug development. The main aim of Paper II was to study galanin and its three receptors in MDD, in our case depressed suicides (Suicides), and controls in five different regions of postmortem brains. We used qPCR to analyze possible changes in expression of transcripts, and pyrosequencing to study DNA methylation. In addition we monitored galanin peptide with radioimmunoassay (RIA). Transcripts for all four members were detected and showed marked regional variations, galanin and GalR1 mRNAs being most abundant. Comparing depressed Suicide subjects and controls, striking results were obtained for galanin and GalR3 transcripts, showing increased mRNA levels, especially in the LC and the DRN of both male and female Suicide subjects, in parallel with decreased DNA methylation. In contrast, galanin and GalR3 transcript levels were decreased in the dorsolateral prefrontal cortex of male Suicide subjects, together with increased DNA methylation, whereas there were no changes in the anterior cingulate cortex. Thus, galanin and its receptor GalR3 are differentially methylated and expressed in brains of MDD subjects in a regionand sex-specific manner. Already previous animal behavioral experiments and human genetic studies suggest that the galanin system is involved in the pathophysiology of mood disorders. The present results further support this view by revealing a novel link between, on one hand, transcriptomic and epigenetic alterations in the galanin system and, on the other hand, suicidal behavior. In Paper III, we examined the distribution patterns of the transcript for another neuropeptide, NPS, and its receptor, in the human brain and compared the results with the localization in the rat brain. Our findings reveal one clear species difference in that the distinct NPS-positive cell cluster seen adjacent to the LC in rat is virtually missing in the human brain. In summary, these results underline putative, important roles of neuropeptides, especially galanin, in normal and pathophysiological conditions. In particular, there are species differences in expression of galanin receptors between human and rodent noradrenergic and serotonergic neurons. Thus, in humans GalR3, surprisingly, seems to be an important receptor in some key brain stem regions and nuclei, like DRN and LC, whereas in rat, instead, GalR1 and GalR2 are abundant receptors. In fact, GalR3 had previously received only modest attention in studies of the rodent brain. These data link changes in mRNA levels and epigenetic alterations in the galanin system to suicidal behavior, making galanin receptors a novel target for development of antidepressant therapeutics. LIST OF SCIENTIFIC PAPERS This thesis is based on the following publications I. Le Maître E, Barde SS, Palkovits M, Diaz-Heijtz R, Hökfelt TG. Distinct features of neurotransmitter systems in the human brain with focus on the galanin system in locus coeruleus and dorsal raphe. Proc Natl Acad Sci USA. 2013 Feb 5; 110(6): E53645. II. Barde SS, Rüegg J, Prud’homme J, Ekström T, Palkovits M, Turecki G, Bagdy G, Ihnatko R, Theodorsson E, Juhasz G, Diaz-Heijtz, Mechawar N, Hökfelt T. Alterations in the galanin system in major depressive disorder: from methylation to peptide. Proc Natl Acad Sci USA, In Press. III. Adori C, Barde S, Bogdanovic N, Uhlén M, Reinscheid RR, Kovacs GG, Hökfelt T. Neuropeptide Sand Neuropeptide S receptor-expressing neuron populations in the human pons. Front Neuroanat. 2015 Sep 25; 9:126. ADDITIONAL PAPERS NOT INCLUDED IN THE THESIS 1. Zhang MD, Barde S, Yang T, Lei B, Eriksson LI, Mathew JP, Andreska T, Akassoglou K, Harkany T, Hökfelt TG, Terrando N. Orthopedic surgery modulates neuropeptides and BDNF expression at the spinal and hippocampal levels. Proc Natl Acad Sci U S A. 2016 Oct 25;113(43):E6686-E6695. 2. Wang P, Li H, Barde S, Zhang MD, Sun J, Wang T, Zhang P, Luo H, Wang Y, Yang Y, Wang C, Svenningsson P, Theodorsson E, Hökfelt TG, Xu ZQ. Depression-like behavior in rat: Involvement of galanin receptor subtype 1 in the ventral periaqueductal gray. Proc Natl Acad Sci U S A. 2016 Aug 9; 113(32):E4726-35. 3. Kawa L, Barde S, Arborelius UP, Theodorsson E, Agoston D, Risling M, Hökfelt T. Expression of galanin and its receptors are perturbed in a rodent model of mild, blast-induced traumatic brain injury. Exp Neurol. 2016 May; 279:159-67. 4. Adori C, Barde S, Vas S, Ebner K, Su J, Svensson C, Mathé AA, Singewald N, Reinscheid RR, Uhlén M, Kultima K, Bagdy G, Hökfelt T. Exploring the role of neuropeptide S in the regulation of arousal: a functional anatomical study. Brain Struct Funct. 2016 Sep; 221(7): 352146. 5. Ádori C, Glück L, Barde S, Yoshitake T, Kovacs GG, Mulder J, Maglóczky Z, Havas L, Bölcskei K, Mitsios N, Uhlén M, Szolcsányi J, Kehr J, Rönnbäck A, Schwartz T, Rehfeld JF, Harkany T, Palkovits M, Schulz S, Hökfelt T. Critical role of somatostatin receptor 2 in the vulnerability of the central noradrenergic system: new aspects on Alzheimer's disease. Acta Neuropathol. 2015 Apr;129(4):541-63. 6. Keimpema E, Zheng K, Barde SS, Berghuis P, Dobszay MB, Schnell R, Mulder J, Luiten PG, Xu ZD, Runesson J, Langel Ü, Lu B, Hökfelt T, Harkany T. GABAergic terminals are a source of galanin to modulate cholinergic neuron development in the neonatal forebrain. Cereb Cortex. 2014 Dec;24(12):3277-88. 7. Mansouri S, Barde S, Ortsäter H, Eweida M, Darsalia V, Langel U, Sjöholm A, Hökfelt T, Patrone C. GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu J Neurochem. 2013 Oct;127(2):209-20.