A role for galanin in human and experimental inflammatory demyelination

Abstract

The neuropeptide galanin is widely expressed by many differing subsets of neurons in the nervous system. There is a marked upregulation in the levels of the peptide in a variety of nerve injury models and in the basal forebrain of humans with Alzheimer's disease. Here we demonstrate that galanin expression is specifically and markedly upregulated in microglia both in multiple sclerosis (MS) lesions and shadow plaques. Galanin expression is also upregulated in the experimental autoimmune encephalomyelitis (EAE) model of MS, although solely in oligodendrocytes. To study whether the observed increase in expression of galanin in inflammatory demyelination might modulate disease activity, we applied the EAE model to a panel of galanin transgenic lines. Over-expression of galanin in transgenic mice (Gal-OE) abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or galanin receptor-2 (GalR2) increase disease severity. The pronounced effects of altered endogenous galanin or GalR2 expression on EAE disease activity may reflect a direct neuroprotective effect of the neuropeptide via activation of GalR2, similar to that previously described in a number of neuronal injury paradigms. Irrespective of the mechanism(s) by which galanin alters EAE disease activity, our findings imply that galanin/GalR2 agonists may have future therapeutic implications for MS.

Fig. 1.

Galanin immunostaining (DAB) of (A) an MS lesion within the parietal lobe white matter, revealing numerous galanin-positive cells, and (B) high power view of a galanin-positive cell in a shadow plaque. In both cases, the ramified morphology of the galanin-positive cells is suggestive of microglia. (C) Double immunofluorescence staining in an MS lesion confirms that the galanin-positive cells (AlexaFluor 488 nm; green) colocalise with the microglial marker CD45 (AlexaFluor 546 nm; red, solid arrow). Of note, not all microglia in MS lesions are galanin-positive (broken arrow). (D) High power image of a galanin and CD45 double-positive cell, with Hoechst nuclear staining (blue). (E) Quantification of galanin-expressing cells in cortical MS lesions, shadow plaques, NAWM and non-MS control tissue, demonstrating 30–50% of MS lesions and shadow plaques are positive for galanin, whilst in contrast <2% of control white matter or NAWM express galanin. Cases C16, C25, MS53, and MS170 were male, and C14, C26, MS120, and MS154 were female.

Fig. 2.

Galanin expression in the mouse spinal cord after EAE. Galanin-expressing cells (green) are indicated by arrowheads in all images. Galanin staining is not observed in (A) GFAP-positive astrocytes (red) or (B) CD11b-positive microglia (red). (C) Many PLPdsRED-positive oligodendrocytes express galanin, but (D) not in naive healthy (no EAE induction) spinal cord from PLPdsRED transgenic mice. (Scale bars, 50 μm.)

Fig. 3.

(A) EAE clinical scores for groups of WT (CBA × C57BL6, n = 10) and Gal-OE (n = 10) mice after immunization with MOG_35–55, demonstrating an absence of disease in the Gal-OE mice, associated with a significant reduction in AUC. (B) EAE clinical scores for groups of WT (129OlaHsd, n = 10) and Gal-KO (n = 10) mice after immunization with MOG_35–55, demonstrating a significant reduction to the time of peak disease in the Gal-KO mice. (C) EAE clinical scores for groups of WT (129SvEvBrd × C57BL6, n = 10) and GalR2-MUT (n = 10) mice after immunization with MOG_35–55, demonstrating a significant reduction in the time of peak disease and a significant increase in disease activity in the GalR2-MUT mice, assessed by AUC. (D) Combined inflammation and demyelination scores for the spinal cord for each genotype and strain-matched WT animals 28 days after the induction of EAE, demonstrating an almost complete absence of disease in the Gal-OE mice and a significant increase in the GalR2-MUT mice. Data are presented as mean ± SEM and statistical significance is denoted by *P < 0.05, ***P < 0.001.

Fig. 4.

Representative histopathological changes in the spinal cord of mice from each of the experimental groups described in Fig. 3, harvested at day 28. Serial longitudinal sections of spinal cord are stained by haematoxylin and eosin (A, C, E, G, I, K) and luxol fast blue (B, D, F, H, J, L). There is an increase is the severity of disease in the GalR2-MUT mice (A and B) compared to strain-matched WT mice (C and D). The pathology is similar in severity and nature in Gal-KO (E and F) compared to strain-matched WT mice (G and H). In each case there is a plaque of granulomatous inflammation over the peripheral spinal cord white matter which focally extends into the underlying parenchyma (marked with an asterisk). The areas of inflammatory change show complete demyelination. Distinct perivascular cuffs of mononuclear inflammatory cells are located deeper in the white matter parenchyma of GalR2-MUT mice (A, marked by arrows). By contrast, the spinal cord from Gal-OE mice (I and J) is histologically normal with no evidence of inflammation or demyelination, compared to strain-matched WT mice (K and L). (Scale bar, 200 μm).