Human Nmnat1 Promotes Autophagic Clearance of Amyloid Plaques in a Drosophila Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by irreversible cognitive decline with limited therapeutic approaches. We characterized a Drosophila model of amyloid pathology that expresses human amyloid-beta precursor protein (APP⁶⁹⁵) and β-site APP cleaving enzyme (BACE) in the nervous system. Our model recapitulates in vivo the age-dependent accumulation of BACE-derived C-terminal fragment (CTF) and amyloid plaques in the brain, one of the key pathological hallmarks of AD. Using this model, we assessed the effects on plaque formation of Nicotinamide mononucleotide adenylyltransferase (Nmnat), an evolutionarily conserved nicotinamide adenine dinucleotide (NAD⁺) synthase involved in cellular metabolism and neuroprotection. We compared the effects of overexpression of Drosophila Nmnat (dNmnat), human Nmnat1 (hNmnat1), human Nmnat2 (hNmnat2), and human Nmnat3 (hNmnat3), and observed that hNmnat1 has the highest efficacy in reducing amyloid aggregation and APP-CTF accumulation. Interestingly, we demonstrated that overexpression of hNmnat1 reduces amyloid plaques by promoting autophagic clearance. Our findings uncover a role of hNmnat1 in amyloid clearance and suggest an exciting neuroprotective potential of hNmnat1 in amyloid pathology.

Keywords: APP; Drosophila; NAD; aggregates; autophagy.

FIGURE 1

Neuronal expression of APP and BACE1 leads to an age-dependent amyloid aggregation in the brain. (A) Western blot analysis showing full-length amyloid precursor protein (APP-FL) and C-terminal fragment (APP-CTF) of 10, 30, and 60 DAE flies expressing APP. Stain-Free imaging of a representative portion of the membrane is presented to show the protein loading. (B) Quantification of CTF/FL ratio in each group. Data are expressed as mean ± SD. n = 5/group. One-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, **P < 0.01. (C) Top row: fly brains at 10, 30, and 60 DAE stained with antibody anti APP (heatmap). Bottom row: higher magnification images of the boxed areas in the top row. Scale bars = 30 μm. (D) Scatter plot showing quantification of the number of amyloid plaques in panel (C). The whiskers represent the minimum and maximum values of the dataset. One-way ANOVA with Bonferroni’s post hoc test. n = 7–8/group, ****P < 0.0001.

FIGURE 2

hNmnat1 reduces the accumulation of amyloid plaques in the brain. (A) Top row: APP staining (heatmap) of brains of flies expressing APP + GFP, APP + dNmnat, APP + hNmnat1, APP + hNmnat2, or APP + hNmnat3 at 60 DAE. Bottom row: higher magnification images of the boxed areas in the top row. Scale bars = 30 μm. (B) Scatter plot showing quantification of the number of amyloid plaques in panel (A). The whiskers represent the minimum and maximum values of the dataset. One-way ANOVA with Bonferroni’s post hoc test. n = 6–8/group, **P < 0.01. (C) Violin plot showing quantification of the size of amyloid plaques in panel (A). One-way ANOVA with Bonferroni’s post hoc test. **P < 0.01. (D) Western blot analysis showing expression of Nmnat isoforms in fly brains at 60 DAE. Actin is used as an internal control. * Indicates a non-specific band.

FIGURE 3

hNmnat1 reduces APP cleavage and production of APP-CTF in the brain. (A) Western-blot analysis of full-length amyloid precursor protein (APP-FL) and C-terminal fragment (APP-CTF) of 10, 30, and 60 DAE flies expressing APP + dNmnat, APP + hNmnat1, APP + hNmnat2, or APP + hNmnat3. Stain-Free imaging of a representative portion of the membrane is presented to show the protein loading. (B) Quantification of CTF/FL ratio in each group. Data are expressed as mean ± SD. n = 4–6/group. One-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, **P < 0.01).

FIGURE 4

hNmnat1 promotes amyloid aggregates clearance through autophagy. (A) Fly brains at 60 DAE were stained with DAPI (blue), APP (magenta), and Atg8 or Ref(2)P (green). Images in the right column are high magnification of the boxed areas (APP is shown in heatmap). Scale bars = 30 μm. (B,C) Quantification of relative Atg8a and Ref(2)P intensity shown in panel (A). Data are expressed as mean ± SD. n = 5/group. Independent sample t-test. *P < 0.05. (D) Western blot analysis showing levels of endogenous Atg8a-I and Atg8a-II in fly brains at 30 and 60 DAE. Actin is used as an internal control. (E,F) Quantification of total Atg8a level (Atg8a-I + Atg8a-II) (E) and the ratio of Atg8a-II to Atg8a-I (F). Data are expressed as mean ± SD. n = 6/group. One-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, **P < 0.01. (G) Western blot analysis showing levels of endogenous Ref(2)P in fly brains at 30 and 60 DAE. Actin is used as an internal control. (H) Quantification of Ref(2)P level. Data are expressed as mean ± SD. n = 6/group. One-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, ****P < 0.0001.