Neuroprotective Role of a Novel Copper Chelator against Aβ 42 Induced Neurotoxicity

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease and associated with the extracellular deposits of amyloid- β peptide in hippocampus region. Metal ions like Cu, Fe and Zn are known to associate with the amyloid beta (A β ) at high concentration and interaction of these ions with soluble and aggregated forms of A β peptide help in development of AD. Here we showed Cu mediated neurotoxicity in the eye tissues of transgenic Drosophila expressing human amyloid β and its rescue through a novel Cu chelator. In this context, we have synthesised and characterized the compound L 2,6-Pyridinedicarboxylic acid, 2,6-bis[2-[(4-carboxyphenyl) methylene] hydrazide] by Mass spectra (MS) and Elemental analysis (EA). The Cu chelation potential of the compound L is tested in vivo in Drosophila. Oral administration of Copper to the transgenic larvae resulted in severe degeneration in eye tissues, which was rescued by the supplementation of compound L. The levels of anti-oxidant markers like SOD and MDA were measured in compound L treated flies and found a significant rescue (P < 0.001). Further rescue of the eye degeneration phenotypes as revealed by SEM affirm the role of copper in A β toxicity. Hence, use of compound L, an amidoamine derivative, could be a possible therapeutic measure for A β induced neurotoxicity.

Figure 1

Crystal structure of compound L (a) and structure of compound L with copper binding (b).

Figure 2

Absorption spectra of compound L = (10⁻⁵M, DMSO-water mixture, 1 : 9 v/v) in the absence and presence of increasing amount of Cu²⁺ = (1–10) × 10⁻⁶ M (in water) at room temperature. Inset of the d-d region appeared at 569 nm after addition of 1 equivalent of Cu(NO₃)₂ solution.

Figure 3

Histogram shows the percentage of flies with normal, mild, and severe eye phenotypes in ey-GAL4 driven UAS-Aβ ₄₂ flies when grown in normal food (a), copper treated food (b), and Cu + chelator treated food (c). The number of flies (n = 100 for (a), (b), and (c)) on y-axes is expressed as % of flies against eye phenotype in each case. Different types of eye degeneration phenotypes of UAS- Aβ ₄₂ /ey-GAL4 like mild ((d) ii) and severe ((d) iii, iv) are shown in panel (d). Note that copper supplemented food resulted in severe ommatidial degeneration as indicated by dark patches in the eye (white arrow in (d) iv).

Figure 4

Histogram shows the percentage of flies having normal, mild, and severe rough eye phenotypes in ey-GAL4 driven UAS-Aβ ₄₂ flies, when grown alone in 200 μM (a), 250 μM (b), and 300 μM (c) of chelator (L) containing food. Note that, at 200 μM, eye degeneration phenotype is rescued as evident by increased number of flies with normal eyes (a). The number of flies in each case is 100.

Figure 5

Measurement of Superoxide dismutase (SOD) in wild type flies and Aβ expressing flies treated in normal food as well as Cu and chelator supplemented food. Data represented are mean ± SD of normal and drug treated groups experiments made in three replicates. Significance is ascribed as *P < 0.05 or **P < 0.001 as compared to wild type.

Figure 6

Measurement of malondialdehyde (MDA) content in wild type flies and Aβ expressing flies treated in normal food as well as Cu and chelator supplemented food. Data represented are mean ± SD of normal and drug treated groups experiments made in triplicates. Significance is ascribed as *P < 0.05 or **P < 0.001 as compared to control.

Figure 7

Scanning electron micrographs showing eye degeneration and their rescue after compound (L) treatment ((e)–(l)). Upper panel shows the digital images of compound eyes of wild type (a), Aβ expressing (b), Aβ in presence of copper (c), and Aβ with Cu + chelator (d). There is a reduction in size of eye of Aβ expressing fly grown in normal food ((b), (f)) as compared to the wild type ((a), (e)) and more degeneration can be seen after treatment with Cu (500 μM) alone ((c), (g)). Rescue after the treatment with 250 μM of compound (L) ((d), (h)). Magnification is 230x. Lower panel shows the eye phenotypes of corresponding images of middle panels, respectively, ((e)–(h)) showing very distinct pattern of eye degeneration and ommatidia disruption. Magnification is 700x.