Mitochondrial DNA damage in a mouse model of Alzheimer's disease decreases amyloid beta plaque formation - PubMed (original) (raw)

Mitochondrial DNA damage in a mouse model of Alzheimer's disease decreases amyloid beta plaque formation

Milena Pinto et al. Neurobiol Aging. 2013 Oct.

Abstract

Mitochondrial DNA (mtDNA) damage and the generation of reactive oxygen species have been associated with and implicated in the development and progression of Alzheimer's disease. To study how mtDNA damage affects reactive oxygen species and amyloid beta (Aβ) pathology in vivo, we generated an Alzheimer's disease mouse model expressing an inducible mitochondrial-targeted endonuclease (Mito-PstI) in the central nervous system. Mito-PstI cleaves mtDNA causing mostly an mtDNA depletion, which leads to a partial oxidative phosphorylation defect when expressed during a short period in adulthood. We found that a mild mitochondrial dysfunction in adult neurons did not exacerbate Aβ accumulation and decreased plaque pathology. Mito-PstI expression altered the cleavage pathway of amyloid precursor protein without increasing oxidative stress in the brain. These data suggest that mtDNA damage is not a primary cause of Aβ accumulation.

Keywords: APP cleavage; Alzheimer; Plaque formation; ROS; mtDNA damage.

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Figures

Fig. 1

The generation of AD-mito-PstI mice to study neuronal mitochondrial DNA (mtDNA) damage in an Alzheimer's disease (AD) background. (A) Breeding scheme used to generate “AD-mito-_PstI_” mice that harbor 3 transgenic alleles. Final experimental animals are “AD+/tTA+/PstI+” and “AD” controls are “AD+/tTA/+” or “AD+/PstI+.” Wild type (wt) controls used for some experiments are age-matched pure C57Bl/6J. (B) Depiction of the suppression and induction scheme to induce mito-PstI expression in an AD background only in adult animals. (C) Representative Western blot analysis detecting the expression of mito-PstI protein in the cortex, hippocampus (hippo), striatum (str), and cerebellum (crbl) of AD-mito-PstI mice. Anti-PstI antibody shows an aspecific band that is indicated by an asterisk (*). Loading was normalized for α-tubulin. (D) Quantification and detection of recombined mtDNA molecules in AD-mito-PstI mice in the cortex, hippo, and str. No deleted mtDNA molecules were detected in AD or wt controls. Each square represents an individual animal (n = 4-5 animals per group). (E) mtDNA copy number is reduced in the cortices of AD-mito-PstI mice compared with AD and wt control mice using qPCR (n = 4-5 animals per group). * p < 0.05. Abbreviations: Dox, doxycycline; E0, embryonic day 0; IRES, internal ribosome entry site; IVS, intervening sequence; nDNA, nuclear DNA.

Fig. 2

Temporal mito-PstI expression leads to a decline in oxidative phosphorylation activity. (A) Representative Western blot analysis probing for different oxidative phosphorylation subunits in cortical, hippocampal, striatal, and cerebellar regions comparing expression between Alzheimer's disease (AD) and AD-mito-PstI mice. (B) Optical density (O.D.) of cytochrome oxidase (Cox) I in cortical homogenates from Western blot analyses comparing AD-mito-PstI and AD mice normalized to a-tubulin (n = 4 per group). (C) Spectrophotometric assay measuring cytochrome oxidase (COX) activity in cortical and hippocampal homogenates of AD and AD-mito-PstI mice (n = 4-5 per group). * p < 0.05. Abbreviations: Atp, adenosine triphosphate subunit; crbl, cerebellum; hippo, hippocampus; Nduf, NADH-ubiquinone oxidoreductase subunit; Sdh, succinate dehydrogenase subunit; str, striatum.

Fig. 3

AD-mito-_Pst_I mice have a significant reduction in the number of amyloid beta (Ab) plaques. (A) Representative images of coronal Alzheimer's disease (AD) and AD-mito-_Pst_I brains immunostained with an anti-Aβ antibody to detect dense and diffuse plaques. Bar = 0.5 mm. (B and C) Quantification of dense and diffuse Aβ plaques in AD and AD-mito-_Pst_I mice in cortex (B) and hippocampus (C) (n = 5-7 per group). Squares represent the quantity of plaques per section from an individual animal. * p < 0.05. (D) Content of Aβ1-42 fragments in cortical and hippocampal homogenates from AD and AD-mito-PstI mice (n = 4/group). Abbreviation: wt, wild type. * p < 0.05.

Fig. 4

Mito-_Pst_I alters Aβ1-42 fragment production but not its degradation. (A) Diagram of the proteolytic processing of APP. The hexagon represents the epitope of the hAPP recognized by 6-10 Ab (only transgenic APPmut), the hexagon combined with the circle represents the epitope of APP recognized by APP-Ct Ab (transgenic and endogenous). (B) Western blot analysis detecting the presence of APPmut in cortical samples from Alzheimer's disease (AD) and AD-mito-_Pst_I mice. (C) Western blot analysis detecting the presence of total APP and β carboxy terminal fragment (CTF) in cortical samples from AD and AD-mito-_Pst_I mice; optical density of APP, b-CTF, and b-CTF/APP signal from Western blot analysis normalized to a-tubulin from cortical samples from AD and AD-mito-_Pst_I mice. * p < 0.05. (D) Enzymatic activity of β-secretase in cortical homogenates of AD and AD-mito-_Pst_I mice (n = 4 per group). (E) Enzymatic activity of IDE in cortical homogenates of AD and AD-mito-_Pst_I mice (n = 5 per group) * p < 0.05. (F) Western blot detecting the presence of 20s proteasome, MMP1, and MMP2 in cortical samples from AD and AD-mito-_Pst_I mice. (G) Western blot detecting the presence of LC3B (I and II) in cortical samples from AD and AD-mito-PstI mice. (H) Optical density (O.D.) of LC3bII/(LC3BI+II) signal from Western blot analysis normalized to a-tubulin from cortical samples from AD and AD-mito-PstI mice. For all the Western blot images: AD, n = 4; AD-mito-PstI, n = 4. Abbreviations: Aβ, antibody; APP, amyloid precursor protein; AICD, amyloid precursor intracellular domain; APPmut, mutant APP; A.U., arbritrary unit; Ct, control; hAPP, human APP; IDE, insulin-degrading enzyme; LC, microtubule-associated protein 1A/1B-light chain; MMP, metalloprotease; sAPP, soluble APP; wt, wild type.

Fig. 5

AD-mito-_Pst_I mice do not show increased reactive oxygen species damage or increased reactive oxygen species production in cortical regions. (A) Rate of hydrogen peroxide production from isolated mitochondria from the cortical regions of Alzheimer's disease (AD) and AD-mito-PstI mice (n = 4 per group). (B) Representative immunohistochemistry with antibody anti- 8-hydroxy-guanosine (8-OHG) and 8-hydroxy-deoxy-guanosine (8-OHdG) on AD and AD-mito-_Pst_I brains (n = 3 mice per group). Scale bar, 200 mm. Negative controls were treated with DNAse and RNAse, positive controls were treated with 30% H2O2. (C) Western blot detecting the presence of 4-hydroxy-2-nonenal (HNE) and SOD2 in cortical samples from AD and AD-mito-_Pst_I mice (AD: n = 3; AD-mito-PstI: n = 4) and relative optical densities (O.D.) from Western blot analyses normalized to a-tubulin. * p < 0.05. (D) Western blot analysis probing for CHOP protein expression in the cortical regions of AD and AD-mito-_Pst_I mice (AD: n = 4, AD-mito-_Pst_I: n = 3). Abbreviations: CHOP, C/EBP homologous protein; SOD, superoxide dismutase.

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Mitochondrial DNA damage in a mouse model of Alzheimer's disease decreases amyloid beta plaque formation - PubMed (original) (raw)