Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

Abstract

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer's disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.

Figure 1. Dendritic spine analysis in Tg2576 mice and wildtype littermates.

(A) Tg2576 animals have deficits in apical dendritic spine density at both four (n = 4) and fourteen (n = 5) months of age, as compared to WT mice (n = 4 at four months; n = 5 at fourteen months), that are reversed by short-term (11 days) PBT2-treatment (n = 3 at both age points). There are also age-related decreases in apical spine density in both the Tg groups. (B) There is a significant increase in basal spine density in fourteen month old Tg animals treated with PBT2. There is also an age-related decrease in basal spine density in WT animals. (C) There were no significant differences in apical or basal dendrite number across the experiment. (D) There were no significant differences in apical or basal dendrite length across the experiment. Values are mean ± SEM. **p<0.01, ***p<0.001.

Figure 2. Biochemical profiles in Tg2576 and wildtype littermate mice and in cell culture.

(A) Tg2576 mice have significant deficits in a number of proteins, as compared to age-matched wildtype controls, and PBT2-treatment significantly increases the expression of a number of these proteins to the levels seen in the control mice. Values have been normalized to the mean of Tg Sham group = 100. (B) The effect of PBT2 on NMDAR1 protein levels in SH-SY5Y cells is abolished in the presence of the high affinity chelator, diamsar (n = 6/treatment). *p<0.05, **p<0.01, #p = 0.06.

Figure 3. The metal-dependent effect of PBT2 on neurite outgrowth.

The effects of copper and zinc alone (0.15 µM) or complexed to PBT2 (0.15 µM) were assessed. (A) PBT2 induces an increase in neurite outgrowth in PC12 cells that is exaggerated when PBT2 is complexed to either copper or zinc (n = 6/treatment). (B) The neuritogenic effect of PBT2 is abolished in the presence of the high affinity chelator, diamsar (n = 9/treatment). Neurite outgrowth in PC12 cells, under control (C), PBT2 (D) and PBT + diamsar (E) conditions is shown in the bottom micrographs (scale bar  = 200 µm). Values are mean ± SEM. **p<0.01, ***p<0.001.