Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology

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.

In vivo Ca2+ imaging of astrocytic microdomains reveals a critical role of the amyloid precursor protein for mitochondria

“…3D‐reconstruction of EM image z ‐stacks revealed shorter and more fragmented, roundish‐shaped mitochondria in APP‐KO astrocytes compared with WT (Figure c,d). This result is in line with previous evidence that the lack of APP results in Ca 2+ and ATP dysregulations (Hamid et al, ; Linde et al, ; Pera et al, ) and morphological alterations of mitochondria (Wang et al, ) suggesting that APP regulates mitochondrial homeostasis.…”

“…The present study additionally reports mitochondria fragmentation in APP‐KO primary astrocyte cultures and suggests that this is resulting from a missing localization of APP to mitochondria where it plays a physiological role. This is in line with a study observing altered mitochondrial morphology (increased proportion of cells exhibiting fragmented mitochondria) and impaired mitochondrial function in HeLa cells that express engineered KPI‐APP variants with decreased mitochondrial localization (Wang et al, ). Wang and colleagues also report increased reactive oxygen species (ROS) in the absence of KPI‐domain in HeLa cell line.…”

“…Moreover, this accumulation of C99 at MAM was shown to interfere with mitochondrial respiratory activity (Pera et al, ). Additionally, the KPI domain of APP, which is predominantly expressed in astrocytes, has been shown to be important for APP localization to mitochondrial (Wang et al, ). More specifically, APP localizes at the translocase of the outer mitochondrial membrane (TOMM) complex, where it affects the import of nuclear encoded proteins into mitochondria (Del Prete et al, ).…”

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

“…129 When A673V and A673T were induced in normal iPSCs by TALEN technology, these cells differentiated and formed cortical neurons, presenting with different levels of AD-associated biomarkers. 130 In addition, through a gene editing platform based on single-stranded oligonucleotide DNA nucleotides and CRISPR/CAS-blocking mutations, Paquet et al 131 generated human iPSCs with dominant ADcausing mutations in APP and presenilin 1 (PSEN1), both heterozygous and homozygous, leading to early disease onset; thereby, they yielded cortical neurons, which showed genotypedependent phenotypes associated with AD. Apolipoprotein E4 (APOE4) is a genetic risk factor for late-onset AD, while ApoE2, which differs from APOE4 by only two bases (two C bases in APOE4, corresponding to two U bases in APOE2), is not a risk factor for AD.…”

Amyloid precursor protein processing and bioenergetics

“…Deletion of this 12 amino acid stretch (which contains a transmembrane domain) decreased APP mitochondrial localization, while also inducing mitochondrial dysfunction. In vitro , this APP deletion mutant led to decreased cell viability, increased ROS, decreased ATP, and depolarized mitochondrial membrane potential (Wang et al, 2016). More studies are warranted to understand the consequences of mitochondrial localized APP.…”