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, 21 (11), 2538-47

Abnormal Interaction Between the Mitochondrial Fission Protein Drp1 and Hyperphosphorylated Tau in Alzheimer's Disease Neurons: Implications for Mitochondrial Dysfunction and Neuronal Damage

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Abnormal Interaction Between the Mitochondrial Fission Protein Drp1 and Hyperphosphorylated Tau in Alzheimer's Disease Neurons: Implications for Mitochondrial Dysfunction and Neuronal Damage

Maria Manczak et al. Hum Mol Genet.

Abstract

We recently reported increased mitochondrial fission and decreased fusion, increased amyloid beta (Aβ) interaction with the mitochondrial fission protein Drp1, increased mitochondrial fragmentation, impaired axonal transport of mitochondria and synaptic degeneration in neurons affected by AD. In the present study, we extended our previous investigations to determine whether phosphorylated tau interacts with Drp1 and to elucidate mitochondrial damage in the progression of AD. We also investigated GTPase activity, which is critical for mitochondrial fragmentation, in postmortem brain tissues from patients with AD and brain tissues from APP, APP/PS1 and 3XTg.AD mice. Using co-immunoprecipitation and immunofluorescence analyses, for the first time, we demonstrated the physical interaction between phosphorylated tau and Drp1. Mitochondrial fission-linked GTPase activity was significantly elevated in the postmortem frontal cortex tissues from AD patients and cortical tissues from APP, APP/PS1 and 3XTg.AD mice. On the basis of these findings, we conclude that Drp1 interacts with Aβ and phosphorylated tau, likely leading to excessive mitochondrial fragmentation, and mitochondrial and synaptic deficiencies, ultimately possibly leading to neuronal damage and cognitive decline. Treatment designed to reduce the expression of Drp1, Aβ and/or phosphorylated tau may decrease the interaction between Drp1 and phosphorylated tau and the interaction between Drp1 and Aβ, conferring protection to neurons from toxic insults of excessive Drp1, Aβ and/or phosphorylated tau.

Figures

Figure 1.
Figure 1.
A representative western blot analysis of normal and phosphorylated tau proteins in brain tissues from early (Braak stages I and II, n = 5), definite (Braak stages III and IV, n = 5) and severe (Braak stages V and VI, n = 5) AD patients, control subjects (Braak stage 0, n = 5) and 3XTg.AD mice (n = 5) and WT mice (n = 5). Normal tau is phosphorylated in AD patients and 3XTg.AD mice.
Figure 2.
Figure 2.
Co-IP analysis of Drp1 and phosphorylated tau in AD patients. (A) Results from IP with a Drp1 antibody and from western blot analysis with a phosphorylated tau antibody. Phosphorylated tau was found in Drp1-IP elutes from AD patients. (B) Western blot analysis of protein lysates from AD patients and control subjects, using the Drp1 antibody.
Figure 3.
Figure 3.
Co-IP analysis of phosphorylated tau and Drp1 in AD patients. (A) IP results with a phosphorylated tau antibody and results from western blot analysis with the Drp1 antibody. Drp1 was found in phosphorylated tau-IP elutes in the cortical tissues from AD patients. (B) Western blots of protein lysates from AD patients and control subjects, using the phosphorylated tau antibody.
Figure 4.
Figure 4.
Co-IP analysis of Drp1 and phosphorylated tau in APP/PS1 and 3XTg.AD mice. (A) IP results with the Drp1 antibody and results from western blot analysis with the phosphorylated tau antibody. Phosphorylated tau was found in Drp1-IP elutes in the cortical tissues from AD patients. (B) Western blot analysis of protein lysates from in APP/PS1 and 3XTg.AD mice, using the Drp1 antibody.
Figure 5.
Figure 5.
Co-IP analysis of phosphorylated tau and Drp1 in APP/PS1 and 3XTg.AD mice. (A) IP results with the phosphorylated tau antibody and results from western blot analysis with the Drp1 antibody. Drp1 was found in phosphorylated tau-IP elutes, in the cortical tissues from AD patients. (B) Results from western blot analysis of protein lysates from APP/PS1 and 3XTg.AD mice, using phosphorylated tau antibody.
Figure 6.
Figure 6.
Double-labeling immunofluorescence analysis of phosphorylated tau and Drp1 in AD patients. The localization of (A) Drp1 and (B) phosphorylated tau, and (C, merged) the colocalization of Drp1 and phosphorylated tau at 40× the original magnification. (D) Images of Drp1, (E) phosphorylated tau and (F) merged at 100× the original magnification. Arrows indicate localization of phosphorylated tau, Drp1 and colocalization of both.
Figure 7.
Figure 7.
Double-labeling immunofluorescence analysis of phosphorylated tau and Drp1 in 3XTg.AD mice. The localization of (A) Drp1 and (B) phosphorylated tau, and (C, merged) the colocalization of Drp1 and phosphorylated tau in the cortex and hippocampus of AD patients; images taken at 40× the original magnification; images of Drp1 and phosphorylated tau were merged at 100× the original magnification. Arrows indicate localization of phosphorylated tau, Drp1 and colocalization of both.
Figure 8.
Figure 8.
GTPase enzymatic activity in cortical tissues of postmortem brains from AD patients—early AD, Braak stages I and II (n = 5), definite AD, Braak stages III and IV (n = 5), severe AD, Braak stage (n = 5) and control subjects (n = 5). GTPase enzymatic activity was significantly decreased in AD patients relative to control subjects.
Figure 9.
Figure 9.
GTPase enzymatic activity in cortical tissues from cortical tissues from the APP (n = 6), APP/PS1 mice (n = 6), 3XTg.AD mice (n = 5) and WT mice (n = 6). GTPase enzymatic activity was significantly increased in the APP, APP/PS1 mice and 3XTg.AD mice relative to WT, non-transgenic mice.

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