Neuroprotective Effects of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) against Amyloid-β-Induced Alzheimer's Disease Mouse Model

Abstract

Alzheimer's disease (AD), is a progressive neurodegenerative disorder that involves the deposition of β-amyloid plaques and the clinical symptoms of confusion, memory loss, and cognitive dysfunction. Despite enormous progress in the field, no curative treatment is available. Therefore, the current study was designed to determine the neuroprotective effects of N-methyl-(2S, 4R)-Trans-4-hydroxy-L-proline (NMP) obtained from Sideroxylon obtusifolium, a Brazilian folk medicine with anti-inflammatory and anti-oxidative properties. Here, for the first time, we explored the neuroprotective role of NMP in the Aβ_1-42-injected mouse model of AD. After acclimatization, a single intracerebroventricular injection of Aβ_1-42 (5 µL/5 min/mouse) in C57BL/6N mice induced significant amyloidogenesis, reactive gliosis, oxidative stress, neuroinflammation, and synaptic and memory deficits. However, an intraperitoneal injection of NMP at a dose of (50 mg/kg/day) for three consecutive weeks remarkably decreased beta secretase1 (BACE-1) and Aβ, activated the astrocyte and microglia expression level as well as downstream inflammatory mediators such as pNF-ĸB, TNF-α, and IL-1β. NPM also strongly attenuated oxidative stress, as evaluated by the expression level of NRF2/HO-1, and synaptic failure, by improving the level of both the presynaptic (SNAP-25 and SYN) and postsynaptic (PSD-95 and SNAP-23) regions of the synapses in the cortexes and hippocampi of the Aβ_1-42-injected mice, contributing to cognitive improvement in AD and improving the behavioral deficits displayed in the Morris water maze and Y-maze. Overall, our data suggest that NMP provides potent multifactorial effects, including the inhibition of amyloid plaques, oxidative stress, neuroinflammation, and cognitive deficits.

Keywords: Alzheimer’s disease; N-methyl-(2S, 4R)-Trans-4-hydroxy-L-proline (NMP); amyloid beta (Aβ1–42); neuroinflammation; neuroprotection; oxidative stress.

Figure 1

(A) Experimental plan for the current study of NMP against Aβ_1–42-induced AD mice model. (B) Chemical structure of N-methyl-(2S, 4R)-Trans-4-hydroxy-L-proline.

Figure 2

The impact of NMP on Alzheimer’s protein indicators within the brains of mice induced with Aβ_1–42. (A) Immunoblot analyses and bar graphs depicting the levels of BACE-1 and Aβ expression in the cortex and hippocampus of mouse brains after the administration of Aβ_1–42 and NMP. (B) Representative images and a corresponding bar graph showing the relative integrated density for Aβ in the cortical and hippocampal tissue (DG region) of mouse brains (n = 4 mice/group). Photomicrograph of (10X) magnification and inset scale bar is 50 µm. Band intensities were measured using ImageJ software (ver. 8.0, San Diego, CA, USA), and the distinctions were illustrated through a bar graph generated by GraphPad Prism. Beta-actin was utilized as a reference for loading. The mean ± S.E.M values for the indicated proteins are displayed as relative integrated density levels (n = 4 mice/group). ** p < 0.01 signify a notable distinction compared to the Aβ_1–42-treated group, while ## p < 0.01 signifies a significant contrast from the vehicle-treated group.

Figure 3

Effects of NMP on astrocytosis and microgliosis within the brains of mice injected with Aβ_1–42. (A) Images of the scanned Western blot results and bar graph for indicated (GFAP and Iba-1) protein expression in the cortex and hippocampus of mice brain following Aβ_1–42 and NMP treatment. The differences are shown in the bar graph. (B) Illustrative images along with an associated bar graph displaying relative integrated density of GFAP in the cortex and hippocampus (DG region) of mouse brains. Photomicrograph of (10X) magnification and inset scale bar is 50 µm. The information is displayed as the average value ± standard error of the mean (n = 4 mice per group). ** p < 0.01 vs. Aβ_1–42-treated group and ## p < 0.01 vs. vehicle-treated group.

Figure 4

Effects of NMP on oxidative stress in the brain of Aβ_1–42-induced mice. (A) Immunoblot analyses and bar graphs depicting the protein expression levels of NRF2 and HO-1 in the cortex and hippocampus of mouse brains after Aβ_1–42 and NMP treatment. (B) Representative photographs and a corresponding bar graph showing relative integrated density of NRF2 in the cortex and hippocampus (DG region) of mouse brains. Photomicrograph of 10X magnification, and inset scale bar is 50 µm. Data are presented as the mean ± S.E.M (n = 4 mice/group). ** p < 0.01 vs. Aβ_1–42-treated group and ## p < 0.01 vs. vehicle-treated group.

Figure 5

Effects of NMP on inflammatory cytokines as well as apoptotic marker in the brain of Aβ_1–42-induced mice. The Western blot assessment and graphical representations indicating the protein expression levels of (pNF-κB, TNF-α, and IL-1β) in the cortexes and hippocampi of mouse brains after Aβ_1–42 and NMP treatment. The bands were assessed and measured utilizing ImageJ software, and the differences are displayed in the bar chart. β-actin served as the standard for loading. The levels of relative density are presented in arbitrary units (A.U.) as the mean ± S.E.M for the specified proteins (n = 4 mice per group). ** p < 0.01 vs. Aβ_1–42-treated group and ## p < 0.01 vs. vehicle-treated group.

Figure 6

Effects of NMP on synaptic proteins in the brain of Aβ_1–42-induced mice. Immunoblot analysis and bar graphs for the cortical and hippocampal protein expression levels of (PSD-95, SNAP-25, SNAP-23, and Synaptophysin) in the brains of mice, followed by Aβ_1–42 and NMP administration. ImageJ software was used for the determination of band densities of these synaptic markers, while the differences are represented by the bar graph that was produced with GraphPad Prism 8 software. The data is displayed as the average ± standard error of the mean (n = 4 mice per group). ** p < 0.01 vs. Aβ_1–42-treated group and ## p < 0.01 vs. vehicle-treated group. β-actin was used as a loading standard.

Figure 7

Effects of NMP on memory impairment and cognitive dysfunction in Aβ_1–42-induced mice. (A) Images of the trajectory map in the MWM and Y-maze task. (B) Line graph showing mean escape latency during training days to reach the visible platform in the MWM task. (C) Time spent in the designated quadrant during the probe trial. (D) Number of crossings around platform during the probe trial. (E) Y-maze task for the measurement of spontaneous alteration behavior percentage in respective groups. The results are shown as the mean ± SEM (n = 8 mice/group). * p < 0.05, and ** p < 0.01 vs. Aβ_1–42-treated group and # p < 0.05, and ## p < 0.01 vs. vehicle-treated group.

Figure 8

Graphical abstract showing the possible neuroprotective effects of NMP in Aβ_1–42-induced mice. An accumulation of amyloid beta (Aβ_1–42) stimulates amyloidogenesis, reactive gliosis, oxidative stress neuroinflammation, and synaptic and memory deficits. These effects are mitigated by NMP in neurodegenerative disorders by reducing the burden of amyloid plaques by reducing the amyloid plaques, gliosis, oxidative stress, as well as neuroinflammation by decreasing the expression level of BACE-1, Aβ, GFAP, Iba-1, ROS, and inflammatory cytokines, while increasing cognitive function by regulating synaptic markers.