Metallotexaphyrins as MRI-Active Catalytic Antioxidants for Neurodegenerative Disease: A Study on Alzheimer's Disease

Abstract

The complex etiology of neurodegeneration continues to stifle efforts to develop effective therapeutics. New agents elucidating key pathways causing neurodegeneration might serve to increase our understanding and potentially lead to improved treatments. Here, we demonstrate that a water-soluble manganese(II) texaphyrin (MMn) is a suitable magnetic resonance imaging (MRI) contrast agent for detecting larger amyloid beta constructs. The imaging potential of MMn was inferred on the basis of in vitro studies and in vivo detection in Alzheimer's disease C. elegans models via MRI and ICP-MS. In vitro antioxidant- and cellular-based assays provide support for the notion that this porphyrin analog shows promise as a therapeutic agent able to mitigate the oxidative and nitrative toxic effects considered causal in neurodegeneration. The present report marks the first elaboration of an MRI-active metalloantioxidant that confers diagnostic and therapeutic benefit in Alzheimer's disease models without conjugation of a radioisotope, targeting moiety, or therapeutic payload.

Keywords: Alzheimer’s disease; C. elegans AD models; MRI; amyloid aggregation; amyloid beta; amyloid beta modifications by ROS and RNS; expanded porphyrin; metalloantioxidant; neurodegeneration; texaphyrin.

Graphical abstract

Figure 1

Chemical Structures of Representative Porphyrinoids As discussed in the text proper, hemin catalyzes oxidative and nitrative damage. Manganese(II) texaphyrin (MMn) is the subject of this study. Manganese(III) tetramethylpyridinium porphyrin (MnTMPyP) is a manganese porphyrin metalloantioxidant. AEOL-10150 has successfully completed a phase I trial in ALS and healthy patients and has shown efficacy in animal models for radiation- and nerve-agent-induced toxicity. Axial ligands are omitted for clarity. See main text for further discussion of these agents.

Figure 2

The Aggregation State of Aβ Peptides as a Function of Incubation Time The three Aβ₄₀ constructs utilized in this study: (left) freshly dissolved peptide is predominantly monomeric in solution, (middle) soluble oligomeric species (partially aggregated) are produced after incubation in solution, and (right) fibrils and fully soluble and insoluble aggregates are formed after prolonged incubation. Aβ₁₆ remains monomeric in solution.

Figure 3

Interaction of MMn with Monomeric and Aggregated Aβ₄₀ (A) Titration with MMn (5 μM) and monomeric Aβ₄₀ with 0 (black), 1 (light blue), and 20 (purple) equiv and (B) photos of aggregated Aβ₄₀ (100 μM; aged 2 months) in the absence and presence of MMn (100 μM) in PBS and after washing with PBS. The pellet shown in the right-most image was obtained by centrifugation and is in methanol.

Figure 4

Modification of Aggregated Aβ₄₀ by MMn as Monitored by CD Spectroscopy and SEM Analyses CD spectra (left) of aggregated (aged for 30 days) Aβ₄₀ (40 μM) were recorded before and after the addition of MMn (20 μM) at t = 0, 7, and 24 h. SEM photographs (right) of (A) Aβ₄₀ aggregate, (B) an Aβ₄₀ aggregate incubated with MMn for 24 h, and (C) monomeric Aβ₄₀ are shown.

Figure 5

In Vitro MRI Analyses of MMn with Various Aβ₄₀ Constructs (A) The longitudinal relaxation rate (R₁) of MMn (140 μM) with monomeric (0.5 equiv, 70 μM), partial (1 week, 0.5 equiv, 70 μM), and aggregated (>2 months) Aβ₄₀ (0.5 equiv, 70 μM) in comparison with MMn alone (140 μM) and in the presence of BSA (0.5 equiv, 70 μM). Dots represent individual samples; the mean (red line) and 95% confidence level for the mean (light red) are also displayed. Standard deviations are either indicated in blue or not shown if within the 95% confidence level. (B) Phantom T1-weighted MRI images of 140 μM MMn were recorded in the presence of increasing concentrations of Aβ₄₀ aggregate (0–70 μM) in PBS at 7 T and 25°C. Samples are normalized to MMn alone, shown in the left-most box.

Figure 6

ICP-MS and In Vivo MRI Analyses of MMn in C. elegans AD Models Mn concentrations (A), as determined by ICP-MS, and (B) MRI analyses of MMn for pellets of wild-type (N2) and AD model (CL4176) C. elegans with and without MMn. Pellets were standardized by dissolving every 1 mg C. elegans in 100 μL HEPES and digesting in HNO₃. n = 5–8 per sample, 2 replicates of each for ICP-MS measurements and n = 2–5 for MRI measurements. Dots represent individual samples; the mean (red line) and 95% confidence level for the mean (light red) are shown. Standard deviation (blue) is shown where larger than the 95% confidence level. *Two-sample t test on ICP-MS variance between MMn-wild-type (WT) and -AD models; p = 0.00018.

Figure 7

¹H NMR Spectroscopic Study of the Hemin-Mediated Oxidation of Serotonin NMR yields were calculated by comparing the spectra of (A) serotonin (1 mM) in pH 7.4 PBS at 37°C in the presence of (B) hemin (15 μM) and an oxidative mixture containing H₂O₂ (1 mM) and sodium ascorbate (40 μM), (C) an H₂O₂-ascorbate oxidative mixture plus MnTMPyP (120 μM), and (D) an H₂O₂-ascorbate oxidative mixture plus MMn (120 μM) recorded after a reaction time of 120 min in all cases.

Figure 8

HPLC-MS Analyses of the Oxidative and Nitrative Modification of Aβ₁₆ (A) Aβ₁₆ incubated under hemin-nitration conditions using heme-H₂O₂-NaNO₂ with varying concentrations of MMn. Percent composition of Aβ₁₆ is shown in black and, as expected, increases with the [MMn]. (B) The protective effect of MMn was also tested against heme-peroxynitrite-mediated modification of Aβ₁₆ in comparison with MnTMPyP. Sodium ascorbate (NaAsc) was added to regenerate the MMn catalyst under the reaction conditions. Reported values are averages with the corresponding standard deviation as obtained from two independent measurements.

Scheme 1

Proposed Mechanism for the MMn-Based Deactivation of Hemin- and Peroxynitrite (ONOO⁻)-Mediated Oxidation and Nitration, as well as Oxidative Decomposition of Hydrogen Peroxide (H₂O₂) Redox cycle of Mn(II/III) texaphyrin. Compounds I and II are generated by the reaction of hemin with H₂O₂. The Aβ peptide and axial ligands are omitted for clarity.

Figure 9

Cellular (Neuro-2A) MTT Assay Neuro-2A cell viability upon exposure to Aβ₄₀ and mixed oxidized-nitrated Aβ₄₀ (hemin-H₂O₂-NaNO₂) with and without MMn, MGd, and MnTMPyP. Reported values are the average with standard deviation of three independent assays each tested four times. The Aβ₄₀ “NO₂” mix is an average of two independent assays each tested four times. *Two-sample t test on percent cell viability (MTT assay) between the Aβ₄₀ “NO₂” mix with and without MMn; p = 0.00016.