Abstract
Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer’s disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer’s neurons.
Highlights
Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/ NAD+) are critical for maintaining redox homeostasis
With 6 hours of an external oxidative shift in Cys/CySS redox state, the intracellular free NADH levels were lowered in mitochondria, cytoplasm and nuclei of all ages, approaching levels needed for survival[13]
We propose that the extracellular reductive Cys/ CySS states enhance the internal free NADH concentrations, but provide substrate energy for enzymatic functions to reverse the energetic deficit in aging and Alzheimer’s disease (AD)
Summary
Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/ NAD+) are critical for maintaining redox homeostasis. We examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. Studies of imposed Cys/CySS redox states demonstrated the feasibility to manipulate internal NAD(P)H levels[12] in mouse neurons and internal glutathione (GSH) levels of human retinal pigment epithelial cells[14]. Since vulnerability to external stress and energetic shortage constitute two of the principal manifestations of aging, here we non-invasively measured free NADH in mitochondria, cytoplasm and nuclei of aged neurons at various redox states. Using fluorescence lifetime imaging microscopy (FLIM), we distinguish the bound and free NADH based on the long and short intrinsic fluorescent lifetimes of NADH16,17 and resolve subcellular compartments for free NADH re-distribution in response to an imposed external oxidative and reductive states in live neurons
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