Mitochondrial dysfunction is associated with familial Alzheimer’s disease (fAD), and the accumulation of damaged mitochondria has been reported as an initial symptom that further contributes to disease progression. In the amyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by β-secretase to generate a C-terminal fragment, which is then cleaved by γ-secretase to produce amyloid-beta (Aβ). The accumulation of Aβ and its detrimental effect on mitochondrial function are well known, yet the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) contributing to this pathology have rarely been reported. We demonstrated the effects of APP-CTFs-related pathology using induced neural stem cells (iNSCs) from AD patient-derived fibroblasts. APP-CTFs accumulation was demonstrated to mainly occur within mitochondrial domains and to be both a cause and a consequence of mitochondrial dysfunction. APP-CTFs accumulation also resulted in mitophagy failure, as validated by increased LC3-II and p62 and inconsistent PTEN-induced kinase 1 (PINK1)/E3 ubiquitin ligase (Parkin) recruitment to mitochondria and failed fusion of mitochondria and lysosomes. The accumulation of APP-CTFs and the causality of impaired mitophagy function were also verified in AD patient-iNSCs. Furthermore, we confirmed this pathological loop in presenilin knockout iNSCs (PSEN KO-iNSCs) because APP-CTFs accumulation is due to γ-secretase blockage and similarly occurs in presenilin-deficient cells. In the present work, we report that the contribution of APP-CTFs accumulation is associated with mitochondrial dysfunction and mitophagy failure in AD patient-iNSCs as well as PSEN KO-iNSCs.

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5′-to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

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