Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer's Disease.

Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer's disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.

Calcium Release Mediated by Redox-Sensitive RyR2 Channels Has a Central Role in Hippocampal Structural Plasticity and Spatial Memory.

AIMS: Previous studies indicate that hippocampal synaptic plasticity and spatial memory processes entail calcium release from intracellular stores mediated by ryanodine receptor (RyR) channels. In particular, RyR-mediated Ca2+ release is central for the dendritic spine remodeling induced by brain-derived neurotrophic factor (BDNF), a neurotrophin that stimulates complex signaling pathways leading to memory-associated protein synthesis and structural plasticity. To examine if upregulation of ryanodine receptor type-2 (RyR2) channels and the spine remodeling induced by BDNF entail reactive oxygen species (ROS) generation, and to test if RyR2 downregulation affects BDNF-induced spine remodeling and spatial memory. RESULTS: Downregulation of RyR2 expression (short hairpin RNA [shRNA]) in primary hippocampal neurons, or inhibition of nitric oxide synthase (NOS) or NADPH oxidase, prevented agonist-mediated RyR-mediated Ca2+ release, whereas BDNF promoted cytoplasmic ROS generation. RyR2 downregulation or inhibitors of N-methyl-d-aspartate (NMDA) receptors, or NOS or of NADPH oxidase type-2 (NOX2) prevented RyR2 upregulation and the spine remodeling induced by BDNF, as did incubation with the antioxidant agent N-acetyl l-cysteine. In addition, intrahippocampal injection of RyR2-directed antisense oligodeoxynucleotides, which caused significant RyR2 downregulation, caused conspicuous defects in a memorized spatial memory task. INNOVATION: The present novel results emphasize the key role of redox-sensitive Ca2+ release mediated by RyR2 channels for hippocampal structural plasticity and spatial memory. CONCLUSION: Based on these combined results, we propose (i) that BDNF-induced RyR2-mediated Ca2+ release and ROS generation via NOS/NOX2 are strictly required for the dendritic spine remodeling and the RyR2 upregulation induced by BDNF, and (ii) that RyR2 channel expression is crucial for spatial memory processes. Antioxid. Redox Signal. 29, 1125-1146.