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, 118 (6), 2190-9

The Autophagy-Related Protein Beclin 1 Shows Reduced Expression in Early Alzheimer Disease and Regulates Amyloid Beta Accumulation in Mice

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The Autophagy-Related Protein Beclin 1 Shows Reduced Expression in Early Alzheimer Disease and Regulates Amyloid Beta Accumulation in Mice

Fiona Pickford et al. J Clin Invest.

Abstract

Autophagy is the principal cellular pathway for degradation of long-lived proteins and organelles and regulates cell fate in response to stress. Recently, autophagy has been implicated in neurodegeneration, but whether it is detrimental or protective remains unclear. Here we report that beclin 1, a protein with a key role in autophagy, was decreased in affected brain regions of patients with Alzheimer disease (AD) early in the disease process. Heterozygous deletion of beclin 1 (Becn1) in mice decreased neuronal autophagy and resulted in neurodegeneration and disruption of lysosomes. In transgenic mice that express human amyloid precursor protein (APP), a model for AD, genetic reduction of Becn1 expression increased intraneuronal amyloid beta (Abeta) accumulation, extracellular Abeta deposition, and neurodegeneration and caused microglial changes and profound neuronal ultrastructural abnormalities. Administration of a lentiviral vector expressing beclin 1 reduced both intracellular and extracellular amyloid pathology in APP transgenic mice. We conclude that beclin 1 deficiency disrupts neuronal autophagy, modulates APP metabolism, and promotes neurodegeneration in mice and that increasing beclin 1 levels may have therapeutic potential in AD.

Figures

Figure 1
Figure 1. Beclin 1 is decreased in AD.
(A and B) Levels of beclin 1 in gray matter from the midfrontal cortex of autopsy-confirmed brains of patients with AD (n = 8), MCI (n = 11), Lewy body variants of AD (LBV; n = 5), HD (n = 7), and age-matched, nondemented, nonpathological controls (C; n = 11). (A) Representative Western blot was probed with anti–beclin 1, anti-NSE, and anti-actin antibodies. (B) Beclin 1 bands were normalized to actin and NSE controls (not shown). Each point represents 1 sample. (C) Ratios of BECN1 mRNA in the entorhinal cortex compared with dentate gyrus in control and AD patients. (D and E) Beclin 1 and APP levels in the cortex of 24-month-old T41 mice (n = 6 APP transgenic, 3 nontransgenic) and 34-month-old J20 mice (n = 5 APP transgenic, 4 nontransgenic). APP expression and Aβ deposition did not reduce beclin 1 levels. Values are mean ± SEM; mean differences were compared by ANOVA and Dunnett’s test (B) or unpaired Student’s t test (C and E).
Figure 2
Figure 2. Beclin 1 and neuronal autophagy are reduced in Becn1+/– mice.
(A and B) Autophagosomes (arrows) were counted in 50 primary hippocampal neurons cultured from GFP-LC3+Becn1+/+ and GFP-LC3+Becn1+/– mice (n = 3 per group). Shown are representative images (A) and quantification (B). (C and D) RIPA buffer–soluble proteins from the cortex of 9-month-old female APP+Becn1+/– and APP+Becn1+/+ mice (n = 7 per group) were analyzed by Western blot and probed for beclin 1; values were normalized to actin levels. (E and F) RIPA buffer–soluble proteins from the cortex of 16-month-old male APP+Becn1+/– and APP+Becn1+/+ mice (n = 4 per group) were analyzed by Western blot and probed for LC3; values were normalized to α-tubulin levels, and the ratio between LC3-I and LC3-II was calculated. Values are mean ± SEM; mean differences were compared by unpaired Student’s t test. Original magnification, ×400.
Figure 3
Figure 3. APP+Becn1+/– mice have increased amyloid pathology.
(A and B) Aβ immunoreactivity in the frontal cortex of 9-month-old female APP+Becn1+/– and APP+Becn1+/+ mice. Coronal brain sections were immunostained with biotinylated anti-Aβ1–5 antibody (3D6). Aβ deposition was defined by the percent area of Aβ immunoreactivity (IR). (C and D) Intracellular Aβ immunoreactivity (4G8) in the hippocampus (Hip) and frontoparietal cortex (Ctx) of 9-month-old female APP+Becn1+/– and APP+Becn1+/+ mice. (E) Total formic acid–soluble Aβ1-x levels measured by ELISA in the neocortex of female APP+Becn1+/– and APP+Becn1+/+ mice. (F) Total formic acid–soluble Aβ1–x levels were inversely correlated with relative beclin 1 protein levels in the cortex. Values are mean ± SEM; mean differences were compared by unpaired Student’s t test. n = 7–8 per genotype. Scale bars: 100 μm (A); 10 μm (C).
Figure 4
Figure 4. Microglial changes in APP+Becn1+/– mice.
(AC) Microglial changes in the frontal cortex of 9-month-old female APP+Becn1+/– and APP+Becn1+/+ mice assessed by immunostaining with antibodies against CD68 and Iba-1. Shown are representative images (A) and quantification (B and C). Arrows denote microglial clusters around amyloid deposits. Values are mean ± SEM; mean differences were compared by unpaired Student’s t test. n = 7–8 per genotype. Scale bar: 50 μm.
Figure 5
Figure 5. Neurodegeneration in 9-month-old APP+Becn1+/– mice.
(AE) Synaptophysin (A and B), MAP-2 (C and D), and calbindin (E) immunoreactivity in 9-month-old female APP+Becn1+/– mice and control littermates. Brain regions analyzed are frontal cortex (A, C, and D), hippocampus (B), and dentate gyrus (E). Coronal brain sections were immunostained with the respective antibodies, and the mean percentage immunoreactive area (AD) or OD (E; diaminobenzidine staining) per mouse was calculated and analyzed by ANOVA and Tukey-Kramer post-hoc test (n = 7–8 per genotype). (A and C) Note the disruption of synaptic and dendritic morphology in APPBecn1+/– and APP+Becn1+/– mice. (F) Neuronal cell counts in layer II of the entorhinal cortex were estimated using the optical dissector method. Results were analyzed by ANOVA and Tukey-Kramer post-hoc test (n = 3 per genotype). Values are mean ± SEM. Original magnification, ×980 (A); ×720 (C).
Figure 6
Figure 6. Abnormal lysosomes in APP+Becn1+/– mice.
Electron micrographs of neurons from 5-month-old mice. APPBecn1+/+ mice had normal lysosomes (single arrows). APP+Becn1+/+ mice had multilaminated dense core bodies in dystrophic neurites (double arrows). APPBecn1+/– mice had lysosomes containing electron-dense bodies (single arrowheads). APP+Becn1+/– mice had abundant enlarged lysosomes containing electron-dense bodies (double arrowheads). Original magnification, ×25,000.
Figure 7
Figure 7. Increasing beclin 1 expression reduces amyloid pathology.
Lentivirus encoding for GFP or mouse beclin 1 (mbeclin 1) was injected into the left (GFP) or right (beclin 1) hippocampus and cortex of 6-month-old APP transgenic mice. (A) GFP and mouse beclin 1 expression 8 weeks after lentivirus injection into the hippocampus. (B and C) Intracellular Aβ (4G8) was reduced with mouse beclin 1 overexpression in the hippocampus (B) and frontoparietal cortex (B and C) of 8-month-old mice. (D) Extracellular β-pleated Aβ deposits measured by thioflavin S staining in the frontal cortex was also reduced by beclin 1 overexpression. Results were analyzed by unpaired Student’s t test (n = 4 per group). Values are mean ± SEM. Original magnification, ×10 (A); ×40 (B).

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