A novel approach to rapidly prevent age-related cognitive decline

Abstract

The loss of cognitive function is a pervasive and often debilitating feature of the aging process for which there are no effective therapeutics. We hypothesized that a novel metal chaperone (PBT2; Prana Biotechnology, Parkville, Victoria, Australia) would enhance cognition in aged rodents. We show here that PBT2 rapidly improves the performance of aged C57Bl/6 mice in the Morris water maze, concomitant with increases in dendritic spine density, hippocampal neuron number and markers of neurogenesis. There were also increased levels of specific glutamate receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate), the glutamate transporter (VGLUT1) and glutamate itself. Markers of synaptic plasticity [calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII, CREB, synaptophysin] were also increased following PBT2 treatment. We also demonstrate that PBT2 treatment results in a subregion-specific increase in hippocampal zinc, which is increasingly recognized as a potent neuromodulator. These data demonstrate that metal chaperones are a novel approach to the treatment of age-related cognitive decline.

Keywords: PBT2; aging; anti-aging; cognition; zinc.

Figure 1

PBT2 improves behavioural outcomes in aged mice. (a) Performance in the learning component of the Morris water maze was assessed over six consecutive days, with mice receiving four trials per day. Data shown are the average escape latencies (seconds). Aged mice (n = 21) were significantly impaired as compared to adult sham-treated animals (n = 7). Acute PBT2 treatment of aged mice (n = 23) resulted in a significant improvement in performance across this task (P = 0.004), with significant differences between the groups emerging on day 3 of testing and persisting for the remainder of the testing period. (b) Performance in the recall task, conducted 24 h after the final learning trial, demonstrated that PBT2 significantly improved the performance of the aged mice (P = 0.0005), which were indistinguishable from adult control animals. (c) Rotarod data, showing the percentage survival of mice on the rod at the different speeds. The PBT2-treated mice (n = 5) performed significantly better (P < 0.05) on this task than the sham-treated controls (n = 5). All data are averages ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

PBT2 improves histological endpoints in aged mice. (a) Golgi analysis was conducted on aged mice (50 neurites per mouse, n = 6 PBT2, n = 5 sham), with PBT2 treatment resulting in a significant increase in both apical and basal spine density. Young control mice (4 months of age, 50 neurites per mouse, n = 3, data not shown) were also analysed, revealing spine density values of 10.4 ± 1.1 (basal) and 12.8 ± 1.3 (apical). PBT2 did not significantly alter apical or basal spine density in young control mice [4 months of age, 50 neurites per mouse, n = 3, data not shown; 10.15 ± 1.3 (basal) and 12.15 ± 1.5 (apical)]. (b) A sample golgi image. Scale bar = 25 μm. (c) Stereological quantitation of the number of Ki67- and doublecortin (DCX)-positive cells in the hippocampus revealed small but significant increases in both following PBT2 treatment (n = 4–6 mice per group with n = 4 sections per mouse). Ki67-positive cells were also significantly increased surrounding the dorsal third ventricle following PBT2 treatment (+214%) in aged animals [n = 5–7 mice per group with n = 4 sections per mouse, P < 0.001 (data not shown)]. (d) Stereological quantitation of the number of NeuN-positive neurons in the various hippocampal subfields revealed a significant increase in neurons in area CA1 and CA3 following PBT2 treatment (n = 4 mice per group with n = 4 sections per mouse). (e) Sample histological images. The first panel shows the entire hippocampus stained for NeuN (scale bar = 250 μm), while the remaining panels show higher power images of NeuN, DCX and Ki67 staining (scale bars = 50 μm). All data are averages ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

PBT2 modulates zinc levels in the hippocampus. (a) A sample laser ablation inductively coupled plasma mass spectrometry (ICPMS) image showing the distribution of zinc in the aged mouse brain, both with and without PBT2 treatment. (b) Quantitation of the laser ablation ICPMS data showing that PBT2 treatment gives rise to an increased level of zinc in area CA1 of the hippocampus, but overall total brain levels are unchanged at this resolution (n = 5–6 per group).

Figure 4

PBT2 modulates biochemical endpoints in the hippocampus. (a) Quantitation of Western blot data (sample composite images shown on the right). The synaptic vesicle zinc transporter, ZnT3, was decreased in the PBT2-treated mice as compared to sham-treated animals. In contrast, the glutamate transporter, VGLUT1, was significantly increased following PBT2 treatment, as compared to sham-treated controls. The levels of other synaptic proteins involved in cognition and plasticity – AMPA and NMDA, PSD95, CREB, CaMKII, phosphorylated CaMKII and synaptophysin (Syn) – were also increased following PBT2 treatment of the aged mice (n = 9–15 per group). (b) Total glutamate levels were also significantly increased in the aged mice following PBT2 treatment (n = 10 per group). (c) The levels of the different protein phosphatase 2A subunits (A, B, C) were also altered following PBT2 treatment (n = 12 per group), as was one of its primary substrates, phosphorylated tau (n = 8 per group) (sample composite images shown on the right). For all Western blot results, data were normalized to GAPDH and then presented as normalized to sham-treated values (100%). All data are averages ± SEM. *P < 0.05, **P < 0.01.