New therapeutic targets in Alzheimer's disease: brain deregulation of calcium and zinc

Abstract

The molecular determinants of Alzheimer's (AD) disease are still not completely known; however, in the past two decades, a large body of evidence has indicated that an important contributing factor for the disease is the development of an unbalanced homeostasis of two signaling cations: calcium (Ca(2+)) and zinc (Zn(2+)). Both ions serve a critical role in the physiological functioning of the central nervous system, but their brain deregulation promotes amyloid-β dysmetabolism as well as tau phosphorylation. AD is also characterized by an altered glutamatergic activation, and glutamate can promote both Ca(2+) and Zn(2+) dyshomeostasis. The two cations can operate synergistically to promote the generation of free radicals that further intracellular Ca(2+) and Zn(2+) rises and set the stage for a self-perpetuating harmful loop. These phenomena can be the initial steps in the pathogenic cascade leading to AD, therefore, therapeutic interventions aiming at preventing Ca(2+) and Zn(2+) dyshomeostasis may offer a great opportunity for disease-modifying strategies.

Figure 1

Aβ, oxidative stress, tau, glutamate receptor overactivation, Ca²⁺, and Zn²⁺ dyshomeostasis may synergistically promote AD-related synaptic and neuronal loss. The sequestration of Zn²⁺ by the amyloid contained in extracellular plaques (1) may remove the cation from the synaptic cleft and release the Zn²⁺-dependent NMDAR blockade (1). Aβ also directly activates Ca-ARs (2). Both phenomena promote Ca²⁺ overload, increased superoxide generation from mitochondria (3), as well as nitric oxide (NO) production from Ca²⁺-dependent activation of NO synthase (NOS) (4). Reactive oxygen and nitrosative species (ROS and RNS) then mobilize Zn²⁺ from MTs leading to toxic Zn²⁺ concentrations (5) that further impair mitochondrial function, promote more ROS generation (6), and release pro-apoptotic factors (7). ROS-driven Zn²⁺ mobilization may also facilitate intra-neuronal Aβ aggregation (8). Aβ may amplify this vicious loop by increasing the generation of ROS that when extracellularly released reduce glutamate reuptake (9) and overactivate NMDARs and Ca-ARs. Mutant PS1 and APP enhance Ca²⁺ dyshomeostasis by altering the ER-mitochondrial network (10). NMDAR-mediated [Ca²⁺]_i rises and oxidative stress may also accelerate tau hyperphosphorylation (11) and recent evidence indicates that oligomers from AD patients are directly promoting tau hyperphosphorylation as well (12). Zn²⁺ sequestration also impairs BDNF signaling (13) (by blocking TrkB and or MMPs), and also reduces the modulating effects exerted by the activation of its own ZnR (14). All these processes, set in motion at the level of synaptic spines, may be the primum movens of synaptic dysfunction, neuronal deafferentation, and death

Figure 2

Glutamate receptor activation: a link between Ca²⁺ and Zn²⁺ dyshomeostasis. NMDAR activation promotes a Ca²⁺ dependent release of intraneuronal Zn²⁺. (a) Neuronal cultures, loaded with the Zn²⁺-sensitive (but Ca²⁺-insensitive) fluorescent probe, FluoZin-3, are shown, before, during, and after a 10 min exposure to NMDA (50 μM), in a Ca²⁺ containing (black) or nominally Ca²⁺ free (red) buffer. (b) Pseudocolor images of FluoZin-3 fluorescent changes upon the experimental condition described in A. Note that the addition of the Zn²⁺ chelator compound, TPEN, completely quenches the FluoZin-3 fluorescence changes, demonstrating the Zn²⁺ dependency of the signal (modified from ref 70)

Figure 3

Zn²⁺ supplementation is beneficial in animal model of AD. (A) Zn²⁺ supplementation prevents the development of hippocampus-dependent memory deficits in 3 × Tg-AD mice. 3 × Tg-AD mice were treated with either water containing 30 p.p.m. of ZnSO₄ or tap water for 11–13 months and tested for the spatial memory version of the MWM. Mice were tested when the platform was removed 1.5 h (to investigate short-term memory) and 24 h (to investigate long-term memory) after the last training trial. Compared with untreated 3 × Tg-AD mice, Zn²⁺-fed 3 × Tg-AD mice did not show any significant difference in their short-term memory performance, but exhibited a marked recovery in their long-term memory as indicated by the decreased time (latency) they employed to reach the point where the platform used to be (Error bars indicate mean values±S.E.M.; ^*P<0.05). (B) Zn²⁺ supplementation reduces both Aβ and tau pathology in the hippocampus of 3 × Tg-AD mice. Immunohistochemistry shows deposits of intraneuronal Aβ (a–d) and h-tau (f–j) in brain slices from treated and untreated 3 × Tg-AD mice (left column: 20 × magnification; right column: 40 × magnification). Compared with untreated mice (a, b), treated 3 × Tg-AD mice showed a significant decrease of intraneuronal Aβ deposits in the hippocampus of (c, d). (b, d) × 40 magnification of the hippocampal CA1 area as shown in the rectangle. (e) Quantification of Aβ load as shown in a and c. Compared with untreated mice (f, g), Zn²⁺-fed 3 × Tg-AD mice showed a strong decrease of intraneuronal h-tau immunoreactivity in the hippocampus of (h, i). (g, i) × 40 magnification of the CA1 area as shown in the rectangle. (j) Quantification of h-tau levels from f and h. Error bars indicate mean values±S.E.M.; ^*P<0.05. (C) Zn²⁺ supplementation promotes MMPs activation in 3 × Tg-AD mice. (a) Gelatine zimography indicates that Zn²⁺ feeding induced a significant increase of MMP-2 and MMP-9 activation in 3 × Tg-AD mice brains. (D). Zn²⁺ supplementation increased brain BDNF levels. BNDF immunoblotting reveals that Zn²⁺-treated 3 × Tg-AD mice showed a fourfold increase in BDNF levels when compared with untreated animals. ^*P<0.05; ^**P<0.01. Error bars indicate mean values±S.E.M. (modified from ref. 79)

Figure 4

Alterations of the ‘zinc set point', implications for AD. The maintenance of brain Zn²⁺ homeostasis is crucial for neuronal functioning. Perturbations of this equilibrium may be a contributing factor in AD development and progression. Excessive Zn²⁺ facilitates Aβ oligomerization as well as overproduction of reactive oxygen species (ROS), thereby promoting synaptic dysfunction and neuronal death. However, Zn²⁺ deficiency may also be deleterious as decreased bioavailability of the cation leads to reduced activation of neuroprotective BDNF signaling. Zn²⁺ deficiency in fact decreases the maturation of pro-BDNF to BDNF and reduces the transactivation of the BNDF receptor, TrkB. Moreover, decreased levels of brain Zn²⁺ can further downregulate the expression and/or the activity of neuroprotective Zn²⁺-dependent proteins that are involved in Aβ clearance (e.g., MMPs) or free radical scavenging (e.g., MTs). Finally, synaptic Zn²⁺deficiency also leads to neurotoxic activation of NMDARs