Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APP SAA knock-in mouse model of Alzheimer's disease.

Introduction: Reduced brain energy metabolism, mTOR dysregulation, and extracellular amyloid-ß oligomer (xcAßO) buildup characterize AD; how they collectively promote neurodegeneration is poorly understood. We previously reported that xcAßOs inhibit N utrient-induced M itochondrial A ctivity (NiMA) in cultured neurons. We now report NiMA disruption in vivo . Methods: Brain energy metabolism and oxygen consumption were recorded in APP SAA/+ mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. Results: NiMA is inhibited in APP SAA/+ mice before other defects are detected in these amyloid-ß-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. GSK3ß signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3ß with lithium or TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APP SAA mice. Conclusion: NiMA disruption in vivo occurs before histopathological changes and cognitive decline in APP SAA mice, and may represent an early stage in human AD.

Detecting RNA-Protein Interactions With EGFP-Cy3 FRET by Acceptor Photobleaching.

Förster Resonance Energy Transfer (FRET) is a great tool for cell biologists to investigate molecular interactions in live specimens. FRET is a distance-dependent phenomenon which can detect molecular interactions at distances between 1-10 nm. Several FRET approaches are reported in the literature for live and fixed cells to study protein-protein interactions; this protocol provides details of acceptor photobleaching as a FRET method to study RNA-Protein interactions. Cy3-labeled RNA foci (FRET acceptors) are photobleached at the intra-cellular site of interest (the nuclei) and the intensity of the EGFP-tagged proteins (FRET donors) at that same site are measured pre- and post- photobleaching. In principle, FRET is detected if the intensity of EGFP increases after photobleaching of Cy3. This protocol describes necessary steps and appropriate controls to conduct FRET measurements by the acceptor photobleaching method. Successful applications of this protocol will provide data to support the conclusion that EGFP-labeled proteins directly interact with Cy3-labeled RNA at the site of photobleaching. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: FRET in fixed cells Alternate Protocol: FRET in live cells.

Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy.

Mitochondria are the central organelles in cellular bio-energetics with key roles to play in energy metabolism and cell fate decisions. Fluorescence Lifetime Imaging microscopy (FLIM) is used to track metabolic changes by following the intrinsic co-enzymes NAD(P)H and FAD, present in metabolic pathways. FLIM records-lifetimes and the relative fractions of free (unbound) and bound states of NAD(P)H and FAD are achieved by multiphoton excitation of a pulsed femto-second infra-red laser. Optimization of multiphoton laser power levels is critical to achieve sufficient photon counts for correct lifetime fitting while avoiding phototoxic effects. We have characterized two photon (2p) laser induced changes at the intra-cellular level, specifically in the mitochondria, where damage was assessed at rising 2p laser average power excitation. Our results show that NAD(P)H-a2%-the lifetime-based enzyme bound fraction, an indicator of mitochondrial OXPHOS activity is increased by rising average power, while inducing changes in the mitochondria at higher power levels, quantified by different probes. Treatment response tracked by means of NAD(P)H-a2% can be confounded by laser-induced damage producing the same effect. Our study demonstrates that 2p-laser power optimization is critical by characterizing changes in the mitochondria at increasing laser average power.

Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay.

Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.

O-Aminobenzoyl-S-nitrosoglutathione: A fluorogenic, cell permeable, pseudo-substrate for S-nitrosoglutathione reductase.

S-nitrosoglutathione reductase (GSNOR) is a multifunctional enzyme. It can catalyze NADH-dependent reduction of S-nitrosoglutathione (GSNO); as well as NAD+-dependent oxidation of hydroxymethylglutathione (HMGSH; an adduct formed by the spontaneous reaction between formaldehyde and glutathione). While initially recognized as the enzyme that is involved in formaldehyde detoxification, increasing amount of evidence has shown that GSNOR also plays a significant role in nitric oxide mediated signaling through its modulation of protein S-nitrosothiol signaling. In humans, GSNOR/S-nitrosothiols have been implicated in the etiology of several diseases including lung cancer, cystic fibrosis, asthma, pulmonary hypertension, and neuronal dysfunction. Currently, it is not possible to monitor the activity of GSNOR in live cells. In this article, we present a new compound, O-aminobenzoyl-S-nitrosoglutathione (OAbz-GSNO), which acts as a fluorogenic pseudo-substrate for GSNOR with an estimated Km value of 320µM. The weak OAbz-GSNO fluorescence increases by approximately 14 fold upon reduction of its S-NO moiety. In live cell imaging studies, OAbz-GSNO is readily taken up by primary pulmonary endothelial cells and localizes to the same perinuclear region as GSNOR. The perinuclear OAbz-GSNO fluorescence increases in a time dependent manner and this increase in fluorescence is abolished by siRNA knockdown of GSNOR or by treatment with GSNOR-specific inhibitors N6022 and C3. Taken together, these data demonstrate that OAbz-GSNO can be used as a tool to monitor the activity of GSNOR in live cells.