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, 20 (13), 2495-509

Impaired Mitochondrial Dynamics and Abnormal Interaction of Amyloid Beta With Mitochondrial Protein Drp1 in Neurons From Patients With Alzheimer's Disease: Implications for Neuronal Damage

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Impaired Mitochondrial Dynamics and Abnormal Interaction of Amyloid Beta With Mitochondrial Protein Drp1 in Neurons From Patients With Alzheimer's Disease: Implications for Neuronal Damage

Maria Manczak et al. Hum Mol Genet.

Abstract

The purpose of our study was to better understand the relationship between mitochondrial structural proteins, particularly dynamin-related protein 1 (Drp1) and amyloid beta (Aβ) in the progression of Alzheimer's disease (AD). Using qRT-PCR and immunoblotting analyses, we measured mRNA and protein levels of mitochondrial structural genes in the frontal cortex of patients with early, definite and severe AD and in control subjects. We also characterized monomeric and oligomeric forms of Aβ in these patients. Using immunoprecipitation/immunoblotting analysis, we investigated the interaction between Aβ and Drp1. Using immunofluorescence analysis, we determined the localization of Drp1 and intraneuronal and oligomeric Aβ in the AD brains and primary hippocampal neurons from Aβ precursor protein (AβPP) transgenic mice. We found increased expression of the mitochondrial fission genes Drp1 and Fis1 (fission 1) and decreased expression of the mitochondrial fusion genes Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), Opa1 (optic atrophy 1) and Tomm40. The matrix gene CypD was up-regulated in AD patients. Results from our qRT-PCR and immunoblotting analyses suggest that abnormal mitochondrial dynamics increase as AD progresses. Immunofluorescence analysis of the Drp1 antibody and the Aβ antibodies 6E10 and A11 revealed the colocalization of Drp1 and Aβ. Drp1 immunoprecipitation/immunoblotting analysis of Aβ antibodies 6E10 and A11 revealed that Drp1 interacts with Aβ monomers and oligomers in AD patients, and these abnormal interactions are increased with disease progression. Primary neurons that were found with accumulated oligomeric Aβ had lost branches and were degenerated, indicating that oligomeric Aβ may cause neuronal degeneration. These findings suggest that in patients with AD, increased production of Aβ and the interaction of Aβ with Drp1 are crucial factors in mitochondrial fragmentation, abnormal mitochondrial dynamics and synaptic damage. Inhibiting, these abnormal interactions may be a therapeutic strategy to reduce mitochondrial fragmentation, neuronal and synaptic damage and cognitive decline in patients with AD.

Figures

Figure 1.
Figure 1.
Immunoblotting analysis of mitochondrial fission, fusion and matrix proteins in early, definite and severe AD patients. Drp1 levels were significantly increased in AD patients at Braak stages I and II (P < 0.005), III and IV (P < 0.02) and V and VI (P < 0.002) compared with the Drp1 levels in control subjects. Fis1 protein levels were also significantly increased [Braak stages I and II (P < 0.003), III and IV (P < 0.01) and IV and V (P < 0.01)] relative to the control subjects. Fusion proteins: in contrast to fission proteins, mitochondrial fusion proteins were significantly decreased in AD patients relative to fission proteins in control subjects. Tomm40 levels were significantly decreased in AD patients at Braak stages IV and V (P < 0.004) relative to the controls. Matrix protein: CypD was significantly increased in AD patients at Braak stages III and IV (P < 0.04) and IV and V (P < 0.02) compared with controls. *Statistical significance at P < 0.05; **statistical significance at P < 0.005.
Figure 2.
Figure 2.
Immunoblotting analysis of Aβ monomers and oligomeric proteins in early, definite and severe AD patients. (A) Immunoblot using 6E10 antibody. Intensity of monomeric and oligomeric Aβ increased in definite and severe AD patients, indicating that Aβ formation and production are dependent on disease progression. Two distinct bands of oligomers (50 and 60 kDa) were found, in addition to two faint bands (25 and 15 kDa; B). Both the 50 kDa (P< 0.001) and the 60 kDa oligomeric Aβ (P< 0.005) were significantly increased in the brains of AD patients at Braak stages I and II relative to the control subjects. (C) Dotblot analysis. (D) Quantification of oligomeric Aβ. *Statistical significance at P< 0.05; **statistical significance at P< 0.005.
Figure 3.
Figure 3.
Immunoprecipitation analysis of Aβ and Drp1. (A) Immunoprecipitation with 6E10 and immunoblotting with 6E10 antibody. 6E10 recognized all derivatives of the mutant AβPP. (B) Immunoprecipitation with the Drp1 monoclonal antibody (Santa Cruz) and immunoblotting with the Drp1 monoclonal antibody.
Figure 4.
Figure 4.
Immunoprecipitation analysis of Aβ and Drp1 antibodies in brain specimens from patients at different stages of AD progression. (A) Immunoprecipitation with Drp1 (polyclonal antibody; Santa Cruz) and immunoblotting with 6E10. 6E10 immunoreacted with full-length AβPP and 4 kDa monomeric Aβ in Drp1, the immunoprecipitated elute from early, definite and severe AD patients, indicating that Drp1 interacts with both full-length AβPP and monomeric Aβ. (B) Immunoprecipitation with Drp1 and immunoblotting with A11. A11 immunoreacted with a band of 50 kDa protein, indicating that Drp1 interacts with oligomeric Aβ.
Figure 5.
Figure 5.
Drp1 localization in the AD brain. (A) Drp1 localization in (a) neurons (red arrows), (b) astrocytes (GFAP) (white arrows) and (c) (merged). Drp1 in (d) neurons (red arrows), (e) microglia (white arrows) and (f) merged. (B) Drp1 expression in neurons from AD patients and control subjects. Levels of Drp1 were higher in neurons from AD patient (b and d) compared with the levels in control subjects (a and c).
Figure 6.
Figure 6.
Aβ in patients with AD. Both intraneuronal and extracellular Aβ were seen in AD patients (A, 5×; B, 10×; and C, 40× original magnifications). Aβ1–40 immunoreactivity and deposits were seen in AD patients (D, 5×; E, 10×; and F, 40× original magnifications). Aβ1–42 immunoreactivity deposits were seen in AD patients (G, 5×; H, 10×; and I, 40× original magnifications).
Figure 7.
Figure 7.
Oligomeric Aβ in neurons from patients with AD. Immunofluorescence analysis of early patients at early stages of AD progression, using A11. [A (40× the original), B and C] The perinuclear accumulation of Aβ oligomers in the AD neurons (100× the original).
Figure 8.
Figure 8.
(A) Double-labeling immunofluorescence analysis of 6E10 and Drp1 in patients with AD. The localization of Drp1 (a) and Aβ (b) and the colocalization of Drp1 and Aβ (c, merged) at 40× the original magnification, and images of Drp1 (d), Aβ (e) and merged (f) at 100× the original magnification. (B) Double-labeling immunofluorescence analysis of A11 and Drp1 in patients with AD. The localization of Drp1 (a) and oligomeric Aβ (b), and the colocalization of Drp1 and Aβ (c, merged) at 40× the original magnification.
Figure 9.
Figure 9.
Immunofluorescence analysis of Aβ in primary hippocampal neurons from AβPP transgenic mice. (A) Aβ (6E10-immunostained) localized in the cell body, axons and nerve terminals in a hippocampal neuron (a). (b, c and d) Aβ localization in hippocampal neuron (100× original magnification). (B) Immunofluorescence analysis of oligomeric Aβ (immunostained with A11) in primary hippocampal neurons from the AβPP transgenic mice. (a) Oligomeric Aβ localization throughout the neuron. (b) Neurons with accumulated oligomeric Aβ, the loss of neuronal projections, damaged neuronal networks and other signs of neurodegeneration.
Figure 10.
Figure 10.
Double-labeling immunofluorescence analysis of Aβ (6E10) and Drp1 in primary neurons from AβPP transgenic mice (A). The localization of Drp1 (a) and 6E10 (b) in hippocampal neurons and the colocalization of Drp1 and Aβ (c, merged) at 100× the original magnification. (B) Double-labeling immunofluorescence analysis of A11 and Drp1 in primary neurons from AβPP transgenic mice. The localization of Drp1 (a) and A11 (b) in hippocampal neuron and the colocalization of Drp1 and Aβ (c, merged) in hippocampal neuron at 100× the original magnification.
Figure 11.
Figure 11.
Double-labeling immunofluorescence analysis of Drp1 and COX1 in primary neurons from AβPP transgenic mice and wild-type mice. Drp1 and COX1 colocalize in cell body, neruites and growth cones. Hippocampal neurons from wild-type or AβPP mice were immunostained for Drp1, mitochondrial marker COX1 and DAPI. Hippocampal neurons from wild-type mice (upper panels) (Drp1, A; COX1, B; DAPI, C; and merged, D) show Drp1 immunoreactivity in growth cones and neurites, whereas neurons from AβPP mice (lower panels) (Drp1, E; COX1, F; DAPI, G; and merged, H) show Drp1 immunoreactivity mainly in the cell body and axons next to the cell body. Areas of active growth were not commonly observed in AβPP cultures. In both wild-type and AβPP cultures Drp1 colocalized with mitochondrial-encoded protein, COX1.

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