Galanin Transgenic Mice with Elevated Circulating Galanin Levels Alleviate Demyelination in a Cuprizone-Induced MS Mouse Model

Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) with a presumed autoimmune etiology. Approved treatments for MS are immunoregulatory and are able to reduce the inflammatory components of the disease. However, these treatments do not suppress progressive clinical disability. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination are likely to improve long-term outcomes and reduce the rate of axonal damage. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system and has diverse neuromodulatory effects. In this study, using the cuprizone (CPZ) demyelination model of MS, we demonstrate that GAL has pronounced neuroprotective effects with respect to demyelination and remyelination. Using our GAL transgenic mouse (GAL-Tg), we identified a novel attenuation of OLs against CPZ induced demyelination, which was exerted independently of progenitor cells. Alleviation of myelin breakdown in the GAL-Tg mice was observed to be significant. Furthermore, we observed changes in the expression of the GAL receptor GalR1 during the demyelination and remyelination processes. Our data strongly indicate that GAL has the capacity to influence the outcome of primary insults that directly target OLs, as opposed to cases where immune activation is the primary pathogenic event. Taken together, these results suggest that GAL is a promising next-generation target for the treatment of MS

Changes in Galanin Systems in a Rat Model of Post-Traumatic Stress Disorder (PTSD).

Post-traumatic stress disorder (PTSD) is a chronic syndrome triggered by exposure to trauma and a failure to recover from a normal negative emotional reaction to traumatic stress. The neurobiology of PTSD and the participation of neuropeptides in the neural systems and circuits that control fear and anxiety are not fully understood. The long-term dysregulation of neuropeptide systems contributes to the development of anxiety disorders, including PTSD. The neuropeptide galanin (Gal) and its receptors participate in anxiety-like and depression-related behaviors via the modulation of neuroendocrine and monoaminergic systems. The objective of this research was to investigate how Gal expression changes in the brain of rats 2 weeks after exposure to footshock. Rats exposed to footshocks were subdivided into high responders (HR; immobility>60%) and low responders (LR; immobility<40%) based on immobility elicited by a novel tone one day after exposure. On day 14, rats were anesthetized, and the amygdala, hypothalamus, pituitary and adrenal glands were removed for analysis using real-time polymerase chain reaction (RT-PCR). Gal mRNA levels were increased in the amygdala and hypothalamus of HR compared with the control and LR. In contrast, Gal mRNA levels were decreased in the adrenal and pituitary glands of HR compared with the control and LR. Thus, the differential regulation (dysregulation) of the neuropeptide Gal in these tissues may contribute to anxiety and PTSD development

Regulation of galanin gene expression in gonadotropin-releasing hormone neurons during the estrous cycle of the rat

Galanin is colocalized with GnRH in neurons of the hypothalamus and basal forebrain of female rats, and this neuropeptide may play a role in the generation of the midcycle surge of gonadotropin secretion. We tested the hypothesis that galanin gene expression in GnRH cells increases during proestrus. To accomplish this, we killed groups of adult female rats at 1200 and 1800 h on the day of proestrus as well as at 1800 h on the day of estrus and used double labeling in situ hybridization and image analysis to estimate and compare the levels of galanin mRNA in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, while the galanin cRNA probe was labeled with 35S and detected by autoradiography. There was no significant difference in the total number of GnRH cells identified in each animal in any of the different groups in any experiment. The relative number of silver grains over these cells, reflecting galanin mRNA content in GnRH neurons (identified by their purple color), was counted with a computerized image analysis system. In an initial experiment, we observed a 2-fold (P < 0.03) higher galanin mRNA signal level in the animals killed at 1800 h than in those killed at 1200 h on the day of proestrus. Animals killed at 1800 h on the day of estrus had galanin mRNA signal levels that were not statistically different from those in the proestrous 1800 h group, indicating that the increase in galanin mRNA at proestrus is maintained for at least 24 h. Galanin mRNA levels in GnRH neurons returned to basal levels equivalent to those in the proestrous 1200 h group by 1000 h on diestrous day 1. In conjunction with the studies of galanin gene expression in GnRH neurons, we compared the relative cellular contents of GnRH mRNA among the same groups. Here, we used single labeling isotopic in situ hybridization for GnRH mRNA and computerized image analysis to count the resulting silver grains. We could detect no difference in GnRH mRNA signal levels (proestrus, 1200 h vs. proestrus, 1800 h vs. estrus, 1800 h). In a final experiment, we investigated the possible role of estrogen in the induction of galanin mRNA expression at proestrus by comparing relative galanin mRNA contents in GnRH neurons among groups of ovariectomized, intact (diestrous day 1), and ovariectomized 17 beta-estradiol-replaced female rats.(ABSTRACT TRUNCATED AT 400 WORDS

The differential expression of GalR1 and GalR2 in the CC area.

<p>RNA samples were extracted from CC areas as demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033901#pone-0033901-g006" target="_blank">Figure 6</a>. The gene expression levels among the groups were normalized to the WT CLT levels (set equal to 1). (A) The expression of GalR1 among the groups. (B) The expression of GalR2 among the groups. Data are expressed as the mean ± SEM values. (n = 3–6 per group). * p<0.05, ** p<0.01.</p

Over-expression of GAL blocks CPZ-induced myelin breakdown.

<p>(A)–(F) are photographs of the cerebral cortex and the CC, while (a)–(d) are the photographs of whole brain. Both Tg and WT mice were given 0.3% CPZ for six weeks (6wCPZ), and then the two groups of mice were allowed to recover for three weeks on a normal food diet (6wCPZ+3wR). Mice brains were processed for IHC staining using an antibody against MBP. Consistent with previous studies, we found extreme demyelination in WT mice after six weeks of CPZ challenge: compare (B) and (b) with the control groups (A) and (a). However, MBP staining of Tg mice brains, (E) and (d), indicated that myelin breakdown was not significantly different compared with controls, (D) and (c). After three weeks on a normal diet, the WT mice recovered well (as expected) (C). To verify the IHC staining results, we also used luxol fast blue staining on WT CLT (I), WT 6wCPZ (II), Tg CLT (III) and Tg 6wCPZ (IV) samples (6 µm paraffin sections). The bar graphs represent the measurements of optical density of MBP IHC staining in the cerebral cortex and the CC areas. Arrows show the aca area that was used for color-intensity standardization. Data are expressed as mean ± s.e.m values. (n = 3–5 per group). ** p<0.01, *** p<0.001.</p

Increased levels of galanin attenuated CPZ-induced oligodendrocyte loss.

<p>Mature oligodendrocytes were detected with IHC using a GST-π antibody. Photographs (A–F) were taken from the knee region of the CC, and (G) and (H) are examples of the full-size pictures taken of WT and Tg brains from the 6wCPZ group. The three CC images in the upper panels (A–C) show the WT mice from the CTL, 6wCPZ and 6wCPZ+3wR groups, and the three images in the middle panels (D–F) show the Tg mice from the same groups. In (A–F), high-magnification micrographs were also taken of the CC area using an oil-immersion lens, as shown in the inserts. (I) A bar chart displaying the numbers of GST-π positive cells, which were counted manually. **p<0.01, ***p<0.001, n = 3 in each group. The longer scale bar represents a length of 200 µm and the shorte scale bar represents 500 µm.</p