Melanocortin receptor agonist NDP-α-MSH improves cognitive deficits and microgliosis but not amyloidosis in advanced stages of AD progression in 5XFAD and 3xTg mice

Abstract

Introduction: Alzheimer's disease (AD) is the most frequent cause of dementia and still lacks effective therapy. Clinical signs of AD include low levels of endogenous melanocortins (MCs) and previous studies have shown that treatment with MC analogs induces neuroprotection in the early stages of AD.

Methods: We investigated the neuroprotective role of MCs in two transgenic mouse models of severe AD using 5 and 7 month-old (mo) 5XFAD mice and 9 and 12 mo 3xTg mice. These mice were subjected to a chronic stimulation of MC receptors (MCRs) with MC analogue Nle4-D-Phe7-α-melanocyte stimulating hormone (NDP-α-MSH, 340 μg/kg, i.p.). Mouse behavior and ex-vivo histological and biochemical analyses were performed after 50 days of treatment.

Results: Our analysis demonstrated an improvement in cognitive abilities of AD mice at late stage of AD progression. We also showed that these protective effects are associated with decreased levels of hyperphosphorylated Tau but not with Aβ burden, that was unaffected in the hippocampus and in the cortex of AD mice. In addition, an age-dependent NDP effect on glial reactivity was observed only in 3xTg mice whereas a global downregulation of p38 mitogen-activated protein kinase was selectively observed in 7 mo 5XFAD and 14 mo 3xTg mice.

Conclusion: Our results suggest that MCR stimulation by NDP-α-MSH could represent a promising therapeutic strategy in managing cognitive decline also at late stage of AD, whereas the effects on neuroinflammation may be restricted to specific stages of AD progression.

Keywords: 3xTg; 5XFAD; NDP-α-MSH; cognitive deficits; neuroprotection.

Figure 1

NDP treatment improves memory in AD mice. A decreased preference for platform searching in the trained target quadrant during the probe test in both 5XFAD (A) and 3xTg (C) is improved by NDP treatment at both experimental ages, indicating that in both AD mouse models the acquisition of the memory for the platform location is impaired; in addition, contrary to 3xTg mice (D), in 5XFAD mice the number of entries in the target quadrant did not significantly change at any experimental age (B). Both parameters were measured in the target quadrant during 60 sec of probe test; data are shown as mean ± SEM. and analyzed by means of one-way ANOVA followed by Bonferroni correction (* vs Wt; ° vs Tg sal; *,°p < 0.05, **, °°p < 0.01, °°°p < 0.001). Experimental groups: B6SJL sal (grey bar): 5 mo: n=17; 7 mo: n=17; 5XFAD sal (black bar): 5 mo: n=21; 7 mo: n=11; 5XFAD NDP (red bar): 5 mo: n=22; 7 mo: n=12; C57BL/6J sal (grey bar): 9 mo: n=6; 14 mo: n=13; 3xTg sal (blue bar): 9 mo: n=7; 14 mo n=20; 3xTg NDP (turquoise bar): 9 mo: n=7; 14 mo: n=20.

Figure 2

Amyloid deposition in 5XFAD mice is not influenced by NDP treatment at any stage of AD progression. Panels (A–H) Representative images showing 6E10+ plaque in CTX (A–D) and DG (E–H) of 5 and 7 mo 5XFAD treated with sal or NDP. Scale bar=200 µm. Panels (I–L) Analysis of 6E10+ plaque area and number in CTX, DG, CA3 and CA1 subfield of Hip of 5 mo and 7 mo of 5XFAD mice treated with sal (black bar) or NDP (red bar). Data are shown as mean ± SEM and were analyzed according to one-way ANOVA; *p < 0.05. Experimental groups: 5XFAD sal: 5 mo: n=10-11; 7 mo: n=6; 5XFAD NDP: 5 mo: n=12; 7 mo: n=6-7. CTX, cortex; DG, dentate gyrus; CA3, cornu Ammonis 3; CA1, cornu Ammonis 1; upMol, upper molecular layer; grDG, granular layer of DG; poDG, polymorph layer of DG; loMol, lower molecular layer.

Figure 3

Amyloid deposition in 3xTg is not influenced by NDP treatment at any stage of AD progression. Panels (A–H) Representative images showing 6E10+ plaque in CA1 (A–D) and SUB (E–H) of 9 and 14 mo 3xTg mice treated with sal or NDP. Scale bar=200 µm. Panels (I–L) Analysis of 6E10+ plaque area and number in CTX, CA3 and CA1 subfield of Hip of 9 mo and 14 mo of 5XFAD mice treated with sal (blue bar) or NDP (turquoise bar). Data are shown as mean ± SEM and were analyzed according to one-way ANOVA. Experimental groups: 3xTg sal: 9 mo: n=6-7; 14 mo n=8; 3xTg NDP: 9 mo: n=6; 14 mo: n=7-8. CTX, cortex; CC, corpus callosum; cg, cingulum; CA3, cornu Ammonis 3; CA1, cornu Ammonis 1; Or, oriens layer; Rad, Radiatum layer; LMol, lacunosum moleculare layer.

Figure 4

NDP treatment does not reduce astrocyte reactivity in the Hip of 5XFAD and 3xTg mice at different experimental ages. Panels A-F; I-N) Representative images showing GFAP ir in DG of 5 (A–C) and 7 (D–F) mo B6SJL and 5XFAD and 9 (I–K) and 14 (L–N) mo C57BL/6J and 3xTg mice treated with sal or NDP. Scale bar=200 µm. Panels (G, H) Analysis of the percentage of area coved by GFAP ir in DG, CA3 and CA1 of Hip of B6SJL (light grey bar) and 5XFAD treated with sal (black bar) or NDP (red bar). (O, P) Percentage of area coved by GFAP ir in DG, CA3 and CA1 and SUB of Hip of C57BL/6J (grey bar) and 3xTg treated with saline (blue bar) or NDP (turquoise bar). Data are shown as mean ± SEM and were analyzed according to one-way ANOVA followed by Bonferroni correction (* vs Wt; ° vs Tg sal; *,°p < 0.05, **p < 0.01, ***p < 0.001). Experimental groups: B6SJL sal: 5mo: n=7; 7 mo: n=8; 5XFAD sal: 5 mo: n=11; 7 mo: n=6; 5XFAD NDP: 5 mo: n=12; 7 mo: n=6-7; C57BL/6J sal: 9 mo: n=4-5; 14 mo: n=6; 3xTg sal: 9 mo: n=7; 14 mo: n=8; 3xTg NDP: 9 mo: n=6; 14 mo: n=7. DG, dentate gyrus; CA3, cornu Ammonis 3; CA1, cornu Ammonis 1; SUB, subiculum; upMol, upper molecular layer; grDG, granular layer of DG; poDG, polymorph layer of DG; loMol, lower molecular layer.

Figure 5

Microglial reactivity in 5XFAD mice is not influenced by NDP treatment at both early and late stage of AD progression. Panels (A–L) Representative images showing IBA1 ir in CTX (A–F) and DG (G–L) of 5 and 7 mo B6SJL control mice treated with saline and 5XFAD mice treated with saline or NDP. Scale bar=200 µm. Panels M-P) Analysis related to the percentage of area coved by IBA1 ir (M, N) and the number of IBA1+ cells (O, P) in CTX, DG, CA3 and CA1 subfields of Hip of 5 mo and 7 mo B6SJL (light grey bar) and 5XFAD mice treated with saline (black bar) or NDP (red bar). Data are shown as mean ± SEM and were analyzed according to one way ANOVA followed by Bonferroni correction (* vs Wt; *p < 0.05, **p < 0.01, ***p < 0.001). Experimental groups: B6SJL sal: 5 mo: n=7; 7 mo=7-8; 5XFAD sal: 5 mo: n=11; 7 mo: n=5; 5XFAD NDP: 5 mo: n=12; 7 mo: n=5. CTX, cortex; DG, dentate gyrus; CA3, cornu Ammonis 3; CA1, cornu Ammonis 1; upMol, upper molecular layer; grDG, granular layer of DG; poDG, polymorph layer of DG; loMol, lower molecular layer.

Figure 6

NDP treatment reduces microglial reactivity in the CA3 of 3xTg mice at early AD stages. Panels (A–L) Representative images showing IBA1 ir in CA1 (A–F) and CA3 (G–L) of 9 and 14 mo C57BL/6J (grey bar) and 3xTg mice treated with sal (blue bar) or NDP (turquoise bar). Scale bar=200 µm. Panels M-P) Analysis of the percentage of area coved by IBA1 ir (M, N) and the number of IBA1+ cells (O, P) in CTX, DG, CA3, CA1 and SUB subfields of Hip of 9 mo and 14 mo C57BL/6J (grey bar) and 3xTg mice treated with sal (blue bar) or NDP (turquoise bar). Data are shown as mean ± SEM and were analyzed according to one-way ANOVA followed by Bonferroni correction (* vs Wt; ° vs Tg sal *,°p<0.05, **p<0.01, ***p < 0.001). Experimental groups: C57BL/6J sal: 9 mo: n=5; 14 mo: n=7; 3xTg sal: 9 mo: n=5-7; 14 mo: n=8; 3xTg NDP: 9 mo: n=5; 14 mo: n=8. CTX, cortex; CC, corpus callosum; DG, dentate gyrus; CA3, cornu Ammonis 3; CA1, cornu Ammonis 1; SUB, subiculum; Or, oriens layer; Rad, Radiatum layer; LMol, lacunosum moleculare layer.

Figure 7

NDP treatment counteracts the expression of AD-related markers in the Hip of 7 mo 5XFAD and 14 mo 3xTg mice. (A) Protein levels measured by western blot analysis in B6SJL (light grey bar) and 5XFAD mice treated with sal (black bar) or NDP (red bar) and (B) in C57BL/6J (grey bar) and 3xTg mice treated with sal (blue bar) or NDP (turquoise bar). Ab-specific optical density (OD) was normalized over ß-Tub signal. Under the graphs representative immunoblots show hippocampal protein levels of ß-Tub, pAPP T688, pTau S202, pTau T181 and p38 MAPK. Data are shown as mean ± SEM and were analyzed according to one-way ANOVA followed by Bonferroni correction (* vs Wt; ° vs Tg sal °p < 0.05, **,°°p < 0.01, ***p < 0.001, ****p < 0.0001). Experimental groups: B6SJL sal: n=10-15; 5XFAD sal: n=8-12; 5XFAD NDP: n=10-15; C57BL/6J sal: n=4; 3xTg 14 mo: n=5; 3xTg NDP: 14 mo: n=5.