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. 2018 Oct;15(4):1036-1054.
doi: 10.1007/s13311-018-0669-5.

L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of Alzheimer's Disease

Baruh Polis  1   2 , Kolluru D Srikanth  3   4 , Evan Elliott  4 , Hava Gil-Henn  3 , Abraham O Samson  5
Affiliations

L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of Alzheimer's Disease

Baruh Polis et al. Neurotherapeutics. 2018 Oct.

Abstract

The urea cycle is strongly implicated in the pathogenesis of Alzheimer's disease (AD). Arginase-I (ARGI) accumulation at sites of amyloid-beta (Aβ) deposition is associated with L-arginine deprivation and neurodegeneration. An interaction between the arginase II (ARGII) and mTOR-ribosomal protein S6 kinase β-1 (S6K1) pathways promotes inflammation and oxidative stress. In this study, we treated triple-transgenic (3×Tg) mice exhibiting increased S6K1 activity and wild-type (WT) mice with L-norvaline, which inhibits both arginase and S6K1. The acquisition of spatial memory was significantly improved in the treated 3×Tg mice, and the improvement was associated with a substantial reduction in microgliosis. In these mice, increases in the density of dendritic spines and expression levels of neuroplasticity-related proteins were followed by a decline in the levels of Aβ toxic oligomeric and fibrillar species in the hippocampus. The findings point to an association of local Aβ-driven and immune-mediated responses with altered L-arginine metabolism, and they suggest that arginase and S6K1 inhibition by L-norvaline may delay the progression of AD.

Keywords: Alzheimer’s disease; L-arginine; L-norvaline; arginase; mTOR.; ribosomal protein S6 kinase β-1.

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Figures

Fig. 1
Fig. 1
Experimental design
Fig. 2
Fig. 2
The effects of L-norvaline on animal behavior. The Y-maze spontaneous alternation test. (a) Percentage of alternation. (b) The total distance traveled during a trial. (c) (MWM) The visible platform test (averaged escape latencies of 2 trials). (d) The hidden platform test (averaged escape latencies of 2 trials/day). (e) The time spent by the mice in the target quadrant in the probe trial. (f) The average swimming speed in the probe test. (g) Heat maps showing the search intensity during the probe trials, where a dashed circle indicates the platform. A high dwell time across the pool area is indicated by colors close to red, whereas colors close to blue indicate lower dwell time (arbitrary scale). Data are shown as means ± SEM. (n = 15 in the non-TG groups, n = 14 in the 3×Tg groups). *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
The impact of L-norvaline on the total amyloid burden. Quantification of the area of Aβ immunoreactivity in brain sections from the 3×Tg mice treated with vehicle (control) or L-norvaline. Representative × 20 magnification view of PFC from the control (a) and L-norvaline-treated mice (b) with apparent deposits in layers IV–V. The insets (c, f) represent × 100 magnification of double staining with DAPI. Intense Aβ deposition in the dentate gyrus (d–f) of the 3×Tg control mice, (f, scale bar 20 μm). Bar chart of the immunoreactive area in the hilus and PFC (g). The Student t test was used to compare the means between 2 groups, *p < 0.05 (n = 15, 3 mice per group)
Fig. 4
Fig. 4
Photomicrographs of Golgi-stained hippocampal neurons from a coronal slice. (a) A representative Golgi-impregnated × 40 image of the CA1 region of non-Tg mice, showing 2 pyramidal cells. The inset is a × 100 image of a secondary apical oblique dendrite in the stratum radiatum. Automated quantitation of spine density using Neurolucida software was subjected to a statistical analysis by a one-way ANOVA. (b) Spine density of hippocampal neurons. *p < 0.05. The data are mean ± SEM, n = 24 for hippocampal cells (3 mice per group). Representative dendritic CA1 segments of 3×Tg control (c) and 3×Tg L-norvaline-treated mice (d). The scale bar is 5 μm
Fig. 5
Fig. 5
L-Norvaline increased the expression levels of neuroplasticity-related proteins in the hippocampus of the 3×Tg mice. Results of the Western blot with β-actin normalized trace quantities (a) and antibody array selected data (b). CFC = change from control
Fig. 6
Fig. 6
L-Norvaline significantly decreased the microglial density in the 3×Tg mice. Visualization and quantification of microglia with Iba1 staining of the hippocampus of the 3×Tg mice (a, b) and non-Tg mice (c, d). Representative hippocampal × 20 bright-field micrographs of the CA3 areas of the control (a, c) and L-norvaline-treated mice (b, d). The boxplots show the area density of microglia in the hippocampal CA3 area of the 3×Tg mice and non-Tg mice treated with vehicle or L-norvaline (e). A significant reduction in the area fraction (%) of Iba1 immunoreactivity in the hippocampal CA3 areas of the 3×Tg mice treated with L-norvaline (f). *p < 0.05 versus controls using a one-way ANOVA with Tukey’s post hoc test, n = 20, 4 mice per group
Fig. 7
Fig. 7
Quantitative characterization of microglial morphology in the CA3 hippocampal area with S = 0.2 mm2. Five sections per mice were included in the analysis. (a) IOD, (b) Feret’s diameter, (c) cell surface area, and (d) sphericity index. A frequency histogram of all microglial cells sampled in the CA3 area with S = 0.2 mm2. Data are presented via box and whisker graphs. The boxes extend from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. The whiskers denote the smallest and largest values. (e) The frequency distribution analysis of the soma area showed a shift from large to smaller cell body sizes after the treatment, and (f) the distribution analysis of circularity indicated that control cells were more likely to possess a more irregular shape (i.e., to have a lower sphericity index)
Fig. 8
Fig. 8
The L-norvaline did not lead to changes in GFAP-positive astroglial density in the 3×Tg mice but led to a significant increase in the volume of somata of astrocytes. Representative hippocampal bright-field × 20 micrographs of the hippocampi from the 3×Tg control (a) and L-norvaline-treated mice (b). The hilus area of the control (c) and treated (d) 3×Tg mice. Representative × 40 images of astrocytes in the CA4 area of the control (e) and treated (f) mice. (g) Bar charts show a significant increase in the area fraction (%) of GFAP-positive cells in the hippocampal hilus area of the 3×Tg mice treated with L-norvaline. (h) GFAP-positive cell density in the hilus. n = 20, 4 mice per group. ***p < 0.001
Fig. 9
Fig. 9
L-Norvaline significantly decreased ARGI immunopositivity in the 3×Tg mice. Representative hippocampal bright-field × 20 micrographs of the control (a) and L-norvaline-treated 3×Tg mice (b). CA4 area of the control (c) and treated (d) 3×Tg mice. CA3 area of the control (e) and treated (f) mice, with × 40 insets (g) and (h), respectively. (i) The boxplots show a significant reduction in the ARGI-positive cell surface area in the CA3 areas (S = 0.2 mm2) and (j) the IOD of ARGI-positive cells in the CA3 area in the 3×Tg mice treated with L-norvaline. Student’s t test (n = 24, with 4 mice per group), **p < 0.01, ***p < 0.001
Fig. 10
Fig. 10
Representative hippocampal CA2–CA3 bright-field × 20 micrographs of the control (a) and L-norvaline-treated 3×Tg mice (b). CA2 × 40 insets of the control (c) and treated (d) mice. (e) A representative × 20 image of the dentate gyrus, with mitochondria stained with ARGII antibody (f) and a × 40 zoomed-in inset clearly showing a mitochondrion. (g) ARGII-positive cell surface area in the CA2 (S = 0.2 mm2). (h) The IOD of ARGII-positive cells in the CA2 area. Student’s t test, *p < 0.05, n = 12, with 4 mice per group
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