Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease.

OBJECTIVES: Patient-derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC-derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of ß-amyloid (Aß) in differentiated neuronal cells. MATERIALS AND METHODS: mtDNA mutations were measured and compared among iPSCs and iPSC-derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aß. RESULTS: Most iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aß deposition. CONCLUSION: This study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aß deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

Modeling of Frontotemporal Dementia Using iPSC Technology.

Frontotemporal dementia (FTD) is caused by the progressive degeneration of the frontal and temporal lobes of the brain. Behavioral variant FTD (bvFTD) is the most common clinical subtype of FTD and pathological subtypes of bvFTD are known as FTD-tau, transactive response (TAR) DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS). Pathological mechanisms of bvFTD are largely unknown. In this study, we investigated the expression of pathological markers, such as p-Tau, TDP-43, and FUS, in the induced pluripotent stem-cell-derived neurons (iPSN) from two sporadic bvFTD patients and one normal subject. We also used an FTD-patient-derived iPSC-line-carrying microtubule-associated protein tau (MAPT) P301L point mutation as positive control for p-Tau expression. Staurosporine (STS) was used to induce cellular stress in order to investigate dynamic cellular responses related to the cell death pathway. As a result, the expression of active caspase-3 was highly increased in the bvFTD-iPSNs compared with control iPSNs in the STS-treated conditions. Other cell-death-related proteins, including Bcl-2-associated X protein (Bax)/Bcl-2 and cytochrome C, were also increased in the bvFTD-iPSNs. Moreover, we observed abnormal expression patterns of TDP-43 and FUS in the bvFTD-iPSNs compared with control iPSNs. We suggest that the iPSC technology might serve as a potential tool to demonstrate neurodegenerative phenotypes of bvFTD, which will be useful for studying pathological mechanisms for FTD as well as related drug screening in the future.

Pathological manifestation of the induced pluripotent stem cell-derived cortical neurons from an early-onset Alzheimer's disease patient carrying a presenilin-1 mutation (S170F).

OBJECTIVES: Alzheimer's disease (AD) is the most common neurodegenerative disease which is characterized by the formation of amyloid beta (Aß) plaques and neurofibrillary tangles. These abnormal proteins induce disturbance in mitochondrial dynamics and defect in autophagy system. Since presenilin-1 (PS1) is a core component in γ-secretase complex, the mutations of PS1 gene cause the interference of γ-secretase activity and lead to the increased Aß42 secretion. We aimed to characterize the patient-specific induced pluripotent stem cell (iPSC) line carrying PS1-S170F mutation. Furthermore, we tested whether disease-modifying drug can reduce AD pathology in the AD iPSC-derived neurons. MATERIALS AND METHODS: Mononuclear cells (MNCs) were isolated freshly from the peripheral blood of an autosomal dominant AD (ADAD) patient carrying presenilin-1 (PS1) mutation (Ser170Phe; PS1-S170F) and a cognitively normal control. We generated induced pluripotent stem cell (iPSC) lines, which were differentiated into functional cortical neurons. Then, we measured the markers indicative of AD pathogenesis using immunocytochemistry and Western blot. We also investigated the mitochondrial dynamics in the AD iPSC-derived neurons using Mito-tracker. RESULTS: We observed that both extracellular and intracellular Aß levels were dramatically increased in the PS1-S170F iPSC-derived neurons, compared with the control iPSC-derived neurons. Furthermore, PS1-S170F iPSC-derived neurons showed high expression levels of p-Tau, which were detected both in the soma and neurites. The mitochondrial velocity in the PS1-S170F iPSC-derived neurons was much reduced, compared with that of the control. We also found a significant decrease of fusion-related protein Mfn1 (membrane proteins mitofusin 1) and an increase of fission-related protein DRP1 (dynamin-related protein 1) in the PS1-S170F iPSC-derived neurons. We further observed the defects of autophagy-related clearance in the PS1-S170F iPSC-derived neurons. Finally, we demonstrated the levels of Aß and p-Tau can be dramatically reduced by the treatment of LY-2886721, a BACE1 inhibitor. CONCLUSIONS: Taken together, we have established and characterized the pathological features of an AD patient carrying PS1-S170F mutation using iPSC technology, which will be the first case on this mutation and this iPSC line will serve as a useful resource for studying AD pathogenesis and drug screening in the future.

The First Generation of iPSC Line from a Korean Alzheimer's Disease Patient Carrying APP-V715M Mutation Exhibits a Distinct Mitochondrial Dysfunction.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease, which is pathologically defined by the accumulation of amyloid plaques and hyper-phosphorylated tau aggregates in the brain. Mitochondrial dysfunction is also a prominent feature in AD, and the extracellular Aß and phosphorylated tau result in the impaired mitochondrial dynamics. In this study, we generated an induced pluripotent stem cell (iPSC) line from an AD patient with amyloid precursor protein (APP) mutation (Val715Met; APP-V715M) for the first time. We demonstrated that both extracellular and intracellular levels of Aß were dramatically increased in the APP-V715M iPSC-derived neurons. Furthermore, the APP-V715M iPSC-derived neurons exhibited high expression levels of phosphorylated tau (AT8), which was also detected in the soma and neurites by immunocytochemistry. We next investigated mitochondrial dynamics in the iPSC-derived neurons using Mito-tracker, which showed a significant decrease of anterograde and retrograde velocity in the APP-V715M iPSC-derived neurons. We also found that as the Aß and tau pathology accumulates, fusion-related protein Mfn1 was decreased, whereas fission-related protein DRP1 was increased in the APP-V715M iPSC-derived neurons, compared with the control group. Taken together, we established the first iPSC line derived from an AD patient carrying APP-V715M mutation and showed that this iPSC-derived neurons exhibited typical AD pathological features, including a distinct mitochondrial dysfunction.