Investigating mitochondrial redox state using NADH and NADPH autofluorescence.

The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues

Heterogeneity and restricted state selection in FRET with fluorescent proteins

Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics

Monitoring cell metabolism with NAD(P)H fluorescence lifetime imaging

In live tissues, alterations in metabolism induce changes in the fluorescence decay of the spectrally identical redox carriers NADH and NADPH. The biochemical pathways and photophysical mechanisms that contribute to these changes are largely unknown. This work combined ultrafast laser spectroscopy and live-cell imaging to investigate these phenomena. Time-resolved spectroscopy of NADH and NADPH was performed using single-photon and two-photon excitation. In solution, the fluorescence lifetimes of the two cofactors were identical. The anisotropy decay dynamics of both molecules indicated that distinct molecular configurations caused the presence of two emitting states, perhaps involving alternate cis/trans geometries of the amide group. Using a range of water/glycerol mixtures as solvents, the viscosity dependence of the non-radiative decay of NAD(P)H was shown to be well described by Kramers and Kramers-Hubbard models of activated barrier crossing. This suggested that variations in the fluorescence lifetimes of the cofactors when bound to different enzymes result from differing levels of conformational restriction of the nicotinamide ring in the binding site. Despite identical fluorescence lifetimes in solution, studies on genetically modified cell lines in which NAD kinase was overexpressed or knocked down indicated that intracellular NADPH was associated with a significantly larger fluorescence lifetime when bound to enzymes (~4.4 ns) than enzyme-bound NADH (~1.5 ns). This suggested that variations in the NAD(P)H fluorescence decay upon metabolic perturbation by pharmacological or pathological means, reported both in this work and in the literature, result from changes in the relative concentrations of NADH and NADPH. NAD(P)H FLIM was used to observe elevated NADPH concentrations in the support cells of the mammalian cochlea, highlighting the potential of the technique as a label-free method for monitoring the metabolic state of complex tissue preparations

Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy

Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ(2) orientation factor arising from differences in donor-acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor-acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET

Polarized Two-Photon Absorption and Heterogeneous Fluorescence Dynamics in NAD(P)H

Two-photon absorption (2PA) finds widespread application in biological systems, which frequently exhibit heterogeneous fluorescence decay dynamics corresponding to multiple species or environments. By combining polarised 2PA with time-resolved fluorescence intensity and anisotropy decay measurements, we show how the two-photon transition tensors for the components of a heterogeneous population can be separately determined, allowing structural differences between the two fluorescent states of the redox cofactor NAD(P)H to be identified. The results support the view that the two states correspond to alternate configurations of the nicotinamide ring, rather than folded and extended conformations of the entire molecule

Decellularised cartilage directs chondrogenic differentiation: creation of a fracture callus mimetic

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularised xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X (Vac-Stx) and SDS (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50ng/mg of tissue, whilst retaining 85% of the sGAG content and as such was selected as the optimal methodology for decellularisation. The resultant Vac-OS scaffolds (dcECM) were also devoid of the immunogenic alpha-gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry (DSC) and fluorescence lifetime imaging microscopy (FLIM). The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2-macrophage driven regenerative response both at 2 and 8 weeks post-implantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1-macrophage mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair

The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α

Triple-negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca(2+) uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca(2+) uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU-silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced, suggesting a signaling role for mROS and HIF-1α, downstream of mitochondrial Ca(2+) Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF-1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca(2+) uptake is a potential novel therapeutic target for clinical intervention

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

A statistical framework for cross-tissue transcriptome-wide association analysis

Transcriptome-wide association analysis is a powerful approach to studying the genetic architecture of complex traits. A key component of this approach is to build a model to impute gene expression levels from genotypes by using samples with matched genotypes and gene expression data in a given tissue. However, it is challenging to develop robust and accurate imputation models with a limited sample size for any single tissue. Here, we first introduce a multi-task learning method to jointly impute gene expression in 44 human tissues. Compared with single-tissue methods, our approach achieved an average of 39% improvement in imputation accuracy and generated effective imputation models for an average of 120% more genes. We describe a summary-statistic-based testing framework that combines multiple single-tissue associations into a powerful metric to quantify the overall gene–trait association. We applied our method, called UTMOST (unified test for molecular signatures), to multiple genome-wide-association results and demonstrate its advantages over single-tissue strategies