Abstract
Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain. Aβ plaques are produced through sequential β/γ cleavage of amyloid precursor protein (APP), of which there are three main APP isoforms: APP695, APP751 and APP770. KPI-APPs (APP751 and APP770) are known to be elevated in AD, but the reason remains unclear. Transcription activator-like (TAL) effector nucleases (TALENs) induce mutations with high efficiency at specific genomic loci, and it is thus possible to knock out specific regions using TALENs. In this study, we designed and expressed TALENs specific for the C-terminus of APP in HeLa cells, in which KPI-APPs are predominantly expressed. The KPI-APP mutants lack a 12-aa region that encompasses a 5-aa trans-membrane (TM) region and 7-aa juxta-membrane (JM) region. The mutated KPI-APPs exhibited decreased mitochondrial localization. In addition, mitochondrial morphology was altered, resulting in an increase in spherical mitochondria in the mutant cells through the disruption of the balance between fission and fusion. Mitochondrial dysfunction, including decreased ATP levels, disrupted mitochondrial membrane potential, increased ROS generation and impaired mitochondrial dehydrogenase activity, was also found. These results suggest that specific regions of KPI-APPs are important for mitochondrial localization and function.
Highlights
Alzheimer’s disease (AD) is a neurodegenerative disorder typically affecting individuals over 65 years of age
To target a specific region of app in the genome, highly active transcription activator-like effector nucleases (TALENs) specific to exon 17 of app were designed (Fig. 1A), and plasmids encoding TALEN binding pairs were transfected into HeLa cells
The PCR-amplified products (1F/1R) covering exon 17 were sequenced, and multiple peaks at the target site suggested that the appTALENs efficiently induced mutations
Summary
Alzheimer’s disease (AD) is a neurodegenerative disorder typically affecting individuals over 65 years of age. In APP695- and APPswe- overexpressing human neuroblastoma cells, DRP1 and OPA1 are significantly decreased, along with fragmented and punctiform mitochondria[11], and overexpression of APP751 significantly decreases the expression levels of mitochondrial metabolic enzymes as well as the mitochondrial membrane potential[18]. These results are based on ectopic overexpression, and the observations might be artifactual. Mutated KPI-APPs show decreased mitochondrial localization, and altered mitochondrial morphology in cells expressing these mutants occurs through disruption of the fission and fusion balance Loss of this region in KPI-APPs induces mitochondrial dysfunction
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