Neuropeptide and Small Transmitter Coexistence: Fundamental Studies and Relevance to Mental Illness

Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanin’s role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression–like behavior. The translational potential of these results was tested by studying the galanin system in postmortem human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a ‘brake’ to prevent overexcitation, a brake that is also part of resilience to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting – a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment

Brain galanin sytems and their role in depression-like behaviour

The aim of the present thesis was to investigate the role of the neuropeptide galanin and galanin receptor subtypes in behavioural functions with a particular focus on depression-like behaviour. In agreement with its wide distribution in the central nervous system, galanin has been implicated in various functions, including regulation of reproduction, endocrine functions, antinociception, attention, learning and memory as well as regulation of mood. Under normal circumstances the levels of galanin expression in several neuronal systems, including dorsal root ganglia, hippocampus and cerebral cortex, are low. However, they may be markedly upregulated following, for example, lesions and/or colchicine treatment. Moreover, in Alzheimer s disease, galanin levels are elevated in the cerebral cortex, hippocampus and basal forebrain. To further analyse the functional roles of galanin in the central and peripheral nervous systems, mice overexpressing galanin (GalOE) under the platelet-derived growth factor B (PDGF-B) promoter were generated and characterised both morphologically and behaviourally. In GalOE mice, a large proportion of sympathetic and sensory ganglia neurons as well as spinal motor neurons were galanin-positive. Expression of galanin in the brain of GalOE mice was more abundant than in wild type controls. A particularly high expression was observed in the olfactory bulb, cerebral cortex, hippocampus and some motor brainstem nuclei. GalOE mice showed an attenuated plasma extravasation in response to mustard oil, an increase in pain response in the formalin test, and changes in muscle physiology. This suggests that overexpressed galanin can be released and influence physiological processes via a receptor-mediated action. Young adult GalOE mice showed a slight increase in the spontaneous locomotor activity, but a decreased response to D-amphetamine treatment. Anxiety-like behaviour as well as spatial and emotional learning and memory were not affected in the GalOE mice. However, GalOE mice displayed an increased time of immobility in the forced swim test, suggesting that an increase in galanin levels may lead to a higher stress-susceptibility evidenced by the development of depression-like behaviour. In support, GalOE mice displayed a significantly higher increase of hippocampal noradrenaline and serotonin release in response to repeated forced swimming as measured by in vivo microdialysis, suggesting that hippocampal afferents of GalOE mice are hypersensitive to stressful stimuli. Intracerebroventricular infusion of galanin to rats resulted in an increased time of immobility in the forced swim test, indicative of depression-like behaviour. This effect was blocked by co-administration of the galanin antagonist M35, while M35 alone caused a decrease of immobility time. These results indicate a potential involvement of galanin in depression-like behaviour and suggest that galanin antagonists may have antidepressant properties. Intracerebroventricular infusion of the galanin receptor GalR1 agonist M617 significantly increased immobility time, similar to galanin itself. In contrast, the GalR2 agonist M1896 decreased, while the GalR2 antagonist M871 increased, time of immobility. These results indicate that galanin receptor subtypes have a differential role in modulation of depression-like behaviour. Galanin may mediate its pro-depressive action via GalR1 subtype, while GalR2 may mediate the putative antidepressant action of galanin. In situ hybridisation studies, performed following exposure to swim stress and galaninergic ligands infusion, showed that the combination of injection and swim stress upregulated tyrosine hydroxylase and galanin mRNA expression in the locus coeruleus, but not tryptophan hydroxylase 2 and galanin mRNA expression in the dorsal raphe. In contrast, following intracerebroventricular galanin infusion, galanin mRNA levels were upregulated both in the locus coeruleus and the dorsal raphe. The levels of the 5-HT1A receptor mRNA were reduced in the dorsal raphe following galanin infusion, and both in the dorsal and median raphe following infusion of the GalR2 agonist M1896. In contrast, serotonin transporter mRNA levels were not affected by any treatment. In summary, these results give evidence for an important role of the neuropeptide galanin in regulation of depressionlike behaviour in rodents. Physiological mechanisms underlying the action of galanin are probably mediated via alterations in neuronal activity of the locus coeruleus and the raphe nuclei, as indicated by changes in gene expression of neuronal markers relevant for noradrenaline, serotonin and galanin transmission. Galanin mechanisms may thus be relevant for mood disorders and galanin ligands may be useful in antidepressant therapy

Galanin expressed in the excitatory fibers attenuates synaptic strength and generalized seizures in the piriform cortex of mice.

The neuropeptide galanin is considered to be an endogenous antiepileptic agent, presumably acting via inhibition of glutamate release. Previously, we have demonstrated that in mice ectopically overexpressing galanin in cortical and hippocampal neurons, particularly in granule cells and their axons, the mossy fibers, hippocampal kindling epileptogenesis is suppressed and is associated with attenuated frequency facilitation in mossy fiber-CA3 cell synapses. We hypothesized that changes in synaptic transmission might occur also in other excitatory synapses of the galanin overexpressing (GalOE) mouse, contributing to seizure suppression. Lateral olfactory tract (LOT) synapses, formed by axons of olfactory bulb (013) mitral cells and targeting piriform cortex (PC) pyramidal cells, ectopically express galanin in GalOE mice. Using whole-cell patch-clamp recordings, we found that excitatory synaptic responses recorded in PC pyramidal cells during high frequency stimulation of the LOT were attenuated in GalOE mice as compared to wild-type controls. This effect was mimicked by bath application of galanin or its agonist galnon to wild-type slices, supporting the notion of ectopic galanin action. Since the high frequency activation induced in vitro resembles epileptic seizures in vivo, we asked whether the observed synaptic inhibition would result in altered epileptogenesis when animals were kindled via the same synapses. In male GalOE mice, we found that the latency to convulsions was prolonged, and once animals had experienced the first stage 5 seizure, generalized seizures were less sustainable. These data indicate that the PC is a possible target for epilepsy treatment by ectopically overexpressing galanin to modulate seizure activity. (c) 2006 Elsevier Inc. All rights reserved

Injection of galanin into the dorsal hippocampus impairs emotional memory independent of 5-HT_1A receptor activation

There is evidence that interaction between the neuropeptide galanin and the 5-HT1A receptor represents an integrative mechanism in the regulation of serotonergic neurotransmission. Thus, in rats intracerebroventricular (i.c.v.) galanin did not impair retention in the passive avoidance (PA) test 24 h after training, but attenuated the retention deficit caused by subcutaneous (s.c.) administration of the 5-HT1A receptor agonist 8-OH-DPAT. This impairment has been linked to postsynaptic 5-HT1A receptor activation. To confirm these results in mice, galanin was infused i.c.v. (1 nmol/mouse) in C57BL/6/Bkl mice 30 min prior to training followed by s.c. injection (0.3 mg/kg) of 8-OH-DPAT or saline 15 min before PA training. In line with previous results, i.c.v. galanin significantly attenuated the PA impairment caused by 5-HT1A receptor activation in mice. To study if the galanin 5-HT1A receptor interaction involved the dorsal hippocampus, galanin (1 nmol/mouse) was directly infused into this brain region alone or in combination with s.c. 8-OH-DPAT. However, unlike i.c.v. galanin, galanin infusion into the dorsal hippocampus alone impaired PA retention and failed to attenuate the 8-OH-DPAT-mediated PA impairment. These results indicate that the ability of i.c.v. galanin to modify 5-HT1A receptor activation is not directly mediated via receptor interactions in the dorsal hippocampus. Instead, the galanin-mediated PA impairment suggests an important inhibitory role of galanin receptors in the dorsal hippocampus for acquisition (encoding) and/or consolidation of emotional memory. In addition, the interaction between galanin and 5-HT1A receptors probably involves a wide serotonergic network that is important for the integration of emotional and cognitive behaviors

SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma.

Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype

Significant association and synergistic adverse prognostic effect of podocalyxin-like protein and epidermal growth factor receptor expression in colorectal cancer

Background: Podocalyxin-like 1 (PODXL) is an anti-adhesive transmembrane protein that has been demonstrated to be an independent factor of poor prognosis in colorectal cancer (CRC). The gene encoding PODXL is located to chromosome 7, which also harbours the gene for the epidermal growth factor receptor (EGFR). The aim of this study was to examine the associations between PODXL and EGFR expression in CRC in vitro and in vivo. Methods: EGFR expression was analysed in tumours from three independent patient cohorts; cohort 1 (n = 533), cohort 2 (n = 259) and cohort 3 (n = 310), previously analysed for immunohistochemical PODXL expression and KRAS and BRAF mutations (cohort 1 and 3). Levels of EGFR and PODXL were determined by western blot in six different CRC cell lines. Results: High expression of PODXL was significantly associated with high EGFR expression (p < 0.001) in all three cohorts, and with BRAF mutation (p < 0.001) in cohort 1 and 3. High EGFR expression correlated with BRAF mutation (p < 0.001) in cohort 1. High EGFR expression was associated with adverse clinicopathological factors and independently predicted a reduced 5-year overall survival (OS) in cohort 1 (HR 1.77; 95 % CI 1.27-2.46), cohort 2 (HR 1.58; 95 % CI 1.05-2.38) and cohort 3 (HR 1.83; 95 % CI 1.19-2.81). The highest risk of death within 5 years was observed in patients with tumours displaying high expression of both EGFR and PODXL in cohort 1 and 3 (HR 1.97; 95 % CI 1.18-3.28 and HR 3.56; 95 % CI 1.75-7.22, respectively). Western blot analysis showed a uniform expression of PODXL and EGFR in all six examined CRC cell lines. Conclusions: The results from this study demonstrate that high expression of EGFR is an independent factor of poor prognosis in CRC. Moreover, strong links have been uncovered between expression of the recently proposed biomarker candidate PODXL with EGFR expression in CRC in vivo and in vitro, and with BRAF mutation in vivo. High expression of both PODXL and EGFR may also have a synergistic adverse effect on survival. These findings suggest a potential functional link in CRC between PODXL, EGFR and BRAF, all originating from chromosome 7, which may be highly relevant in the clinical setting and therefore merit future in-depth study

MOESM1 of Significant association and synergistic adverse prognostic effect of podocalyxin-like protein and epidermal growth factor receptor expression in colorectal cancer

Additional file 1. Patient and tumour characteristics in the three cohorts