Abstract Background Heterozygous mutations in the glucocerebrosidase gene (GBA1), which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are a genetic risk factor for Parkinson’s disease (PD). The pathophysiological consequences of GBA1 mutations on dopaminergic neuronal function, especially their impact on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear. Methods Fibroblasts and dopaminergic neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD were used in the study. Live-cell imaging was performed to measure lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial morphology and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was quantified using fluorescence lifetime imaging with Förster resonance energy transfer (FLIM-FRET), and pharmacological interventions included rapamycin and acidic nanoparticles. Results GCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired in GBA1 mutant dopaminergic neurons. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. Mechanistic target of rapamycin complex 1 (MTORC1) was constitutively phosphorylated and FLIM-FRET measurements confirmed impairment of lysosomal V-ATPase assembly, which was reversed by rapamycin treatment. Rapamycin and lysosome-targeting acidic nanoparticles rescued lysosomal pH and restored mitophagy, mitochondrial membrane potential and mitochondrial oxidative phosphorylation complex level in the GBA1 mutant dopaminergic neurons. Conclusions We revealed a novel mechanistic link between GBA1 mutations and mitochondrial dysfunction, as the disruption of V-ATPase assembly driven by MTORC1 activation impairs lysosomal acidification. This causes impairment of mitophagy, leading to mitochondrial dysfunction, undermining dopaminergic cell function and fate. Pharmacological intervention with rapamycin or acidic nanoparticles restores lysosomal pH and rescue mitochondrial function, representing a novel therapeutic approach for GBA1-PD .

When probed in living systems using fluorescence lifetime imaging microscopy (FLIM), the emission from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form NADPH have shown promise as sensitive intrinsic reporters of metabolism. However, an incomplete understanding of the biochemical processes controlling their fluorescence decay makes it difficult to draw unambiguous conclusions. Here, we utilised time-resolved fluorescence anisotropy imaging to identify multiple enzyme binding configurations associated with lifetimes both longer and shorter than unbound NAD(P)H. FLIM, combined with mathematical and computational modelling, revealed that the redox states of the NAD and NADP pools control the steady-state equilibrium of binding configurations, which in turn determines the observed fluorescence decay. This knowledge will be foundational to developing the accurate interpretation of NAD(P)H FLIM.

NADPH-P450 oxidoreductase (POR) is the accepted redox-partner for drug-metabolising cytochrome P450s (CYPs) over NADH-cytochrome b5 reductase (Cytb5R). Accordingly, POR-centric NADPH-supplemented human liver microsomes (HLM) and recombinant CYP-POR systems complement human hepatocytes (HH) as drug-metabolism models. However, HH may exhibit inexplicably lower CYP activities relative to NADPH-HLM, particularly for CYP3A4-substrates. Here, we show the phenomenon can manifest as disparate CYP phenotyping in which NADPH-HLM and recombinant CYP-POR systems incorrectly identify CYP3A4 while HH accurately assign the main-metabolising CYP exemplified by CYP1A2 for savolitinib. Mechanistically, we serendipitously discover that HH CYP3A4-mediated midazolam-metabolism is increased by gefitinib and find that this can only be recapitulated in non-canonical Cytb5R-dependent NADH-HLM and recombinant CYP3A4-Cytb5R. We conclude that Cytb5R is important for HH-CYP3A4 and show HH-consistent CYP3A4-activities in NADH-HLM. Imaging NADH/NADPH in hepatocytes shows equivalent concentrations suggesting CYP redox-partnerships are cofactor-independent and likely influenced by protein-protein interactions as mimicking the dense-intracellular protein using albumin recapitulates HH savolitinib-metabolism in HLM.

Pathological mutations in liquid-liquid phase separation (LLPS) scaffold proteins have been linked to biomolecular condensate dysfunction in neurodegenerative diseases, while the possible impact of client protein mutations remains unclear. In spinal muscular atrophy (SMA), the disease-associated E134K mutation in the Tudor domain (TD) of the survival motor neuron (SMN) protein disrupts its interaction with GAR1, an RGG-rich protein within the H/ACA small nucleolar ribonucleoprotein complex. The consequences of this impaired interaction have not been elucidated. Here, we identified GAR1 as an LLPS scaffold protein that is phase separated in nuclear compartments and forms gel-like condensates via complex coacervation with RNA in vitro. In cells, we reveal that SMN co-localizes with GAR1 in Cajal bodies and modulates its dynamics. Using confocal microscopy and NMR spectroscopy, we further show that SMN TD is a client protein that regulates the architecture and dynamics of GAR1 condensates through competitive RNA interactions, implicating GAR1 phase separation in RNA accumulation and release processes regulated by SMN. Notably, the SMA-associated E134K variant of SMN exhibits a reduced affinity for GAR1, impairing the modulation of GAR1 condensates and displacement RNA. Our findings suggest a mechanistic link between phase separation dysregulation and SMA, driven by disrupted scaffold-client interactions, that highlights the therapeutic potential of targeting SMN-dependent condensate regulation in SMA. ### Competing Interest Statement The authors have declared no competing interest.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

The transcription factor Nrf2 and its repressor Keap1 mediate cell stress adaptation by inducing expression of genes regulating cellular detoxification, antioxidant defence and energy metabolism. Energy production and antioxidant defence employ NADH and NADPH respectively as essential metabolic cofactors; both are generated in distinct pathways of glucose metabolism, and both pathways are enhanced by Nrf2 activation. Here, we examined the role of Nrf2 on glucose distribution and the interrelation between NADH production in energy metabolism and NADPH homeostasis using glio-neuronal cultures isolated from wild-type, Nrf2-knockout and Keap1-knockdown mice. Employing advanced microscopy imaging of single live cells, including multiphoton fluorescence lifetime imaging microscopy (FLIM) to discriminate between NADH and NADPH, we found that Nrf2 activation increases glucose uptake into neurons and astrocytes. Glucose consumption is prioritized in brain cells for mitochondrial NADH and energy production, with a smaller contribution to NADPH synthesis in the pentose phosphate pathway for redox reactions. As Nrf2 is suppressed during neuronal development, this strategy leaves neurons reliant on astrocytic Nrf2 to maintain redox balance and energy homeostasis.

NADH and NADPH play key roles in the regulation of metabolism. Their endogenous fluorescence is sensitive to enzyme binding, allowing changes in cellular metabolic state to be determined using fluorescence lifetime imaging microscopy (FLIM). However, to fully uncover the underlying biochemistry, the relationships between their fluorescence and binding dynamics require greater understanding. Here we accomplish this through time- and polarization-resolved fluorescence and polarized two-photon absorption measurements. Two lifetimes result from binding of both NADH to lactate dehydrogenase and NADPH to isocitrate dehydrogenase. The composite fluorescence anisotropy indicates the shorter (1.3-1.6 ns) decay component to be accompanied by local motion of the nicotinamide ring, pointing to attachment solely via the adenine moiety. For the longer lifetime (3.2-4.4 ns), the nicotinamide conformational freedom is found to be fully restricted. As full and partial nicotinamide binding are recognized steps in dehydrogenase catalysis, our results unify photophysical, structural, and functional aspects of NADH and NADPH binding and clarify the biochemical processes that underlie their contrasting intracellular lifetimes.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along the basal-to-apical long (tonotopic) axis. To generate the frequency-specific characteristics required at the appropriate positions, nascent HCs must determine their tonotopic properties during development. This relies on complex signalling between the developing HC and its local niche along the axis within the auditory organ. Here we apply NAD(P)H fluorescence lifetime imaging (FLIM) and live imaging of mitochondria to reveal metabolic gradients along the tonotopic axis of the developing chick cochlea. We further show that re-shaping these gradients during development, by inhibiting cytosolic glucose metabolism, alters normal Bmp7 and Chordin like-1 signalling leading to flattening of tonotopic patterning along the axis. Our work supports a causal link between morphogen signalling and metabolic reprogramming in specifying tonotopic morphologies in auditory HCs.### Competing Interest StatementThe authors have declared no competing interest.

The cellular localisation of diphenylacetylene fluorophores can be influenced by modifying their structure, modulating lipophilicity and incorporating ionisable groups.

An increasing volume of data suggests that changes in cellular metabolism have a major impact on the health of tissues and organs, including in the auditory system where metabolic alterations are implicated in both age-related and noise-induced hearing loss. However, the difficulty of access and the complex cyto-architecture of the organ of Corti has made interrogating the individual metabolic states of the diverse cell types present a major challenge. Multiphoton fluorescence lifetime imaging microscopy (FLIM) allows label-free measurements of the biochemical status of the intrinsically fluorescent metabolic cofactors NADH and NADPH with subcellular spatial resolution. However, the interpretation of NAD(P)H FLIM measurements in terms of the metabolic state of the sample are not completely understood. We have used this technique to explore changes in metabolism associated with hearing onset and with acquired (age-related and noise-induced) hearing loss. We show that these conditions are associated with altered NAD(P)H fluorescence lifetimes, use a simple cell model to confirm an inverse relationship between τ bound and oxidative stress, and propose such changes as a potential index of oxidative stress applicable to all mammalian cell types.

Drug-like, donor–acceptor diphenylacetylenes cause efficient cell death upon photoactivation and hence have potential phototherapeutic applications.

Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.

BACKGROUND: Mitochondrial metabolism involves oxidative phosphorylation (OXPHOS) via the respiratory chain and is required for the maintenance of tumour cell proliferation and regulation of epithelial/mesenchymal transition (EMT) related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study we interrogate the role of CHCHD4 regulated respiratory chain activity and metabolism in tumour cell proliferation and EMT-related phenotypes.RESULTS: We show that CHCHD4 is essential for the proliferation of tumour cells irrespective of their aetiology. In human tumours, elevated CHCHD4 expression is correlated with a mitochondrial OXPHOS gene signature and with a proliferative gene signature associated with the mTORC1 signalling pathway. Elevated CHCHD4 increases tumour cell proliferation, in a manner that is dependent on complex I (CI) activity, glutamine consumption and mTORC1 activation. CHCHD4 expression is inversely correlated with EMT gene expression both in vitro and in vivo. Finally, we show CHCHD4 regulates the intracellular distribution of the EMT marker vimentin, in a CI mediated manner.CONCLUSIONS: CHCHD4 regulates tumour cell proliferation and metastatic (EMT related) phenotypes through its control of CI-mediated mitochondrial metabolism.

BackgroundMitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelialtomesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved.ResultsUsing in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway.We show that CHCHD4 expression significantly correlates with thedoubling time of a range of tumour celllines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupledto respiratory chaincomplex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linkedto CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes.ConclusionsCHCHD4 drives tumour cell growthand activates mTORC1 signalling through its control of respiratory chainmediated metabolism and complex I biology, and also regulates EMT-related phenotypes oftumour 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.

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Department of Chemistry, Durham Unive Durham DH1 3LE, UK. E-mail: andy.whitin Department of Biosciences, Durham Univer Biophysical Sciences Institute, Departmen Road, Durham, DH1 3LE, UK LightOx Limited, Wynyard Park House, Wyn 5TB, UK Bioscience Technology Facility, Department 5DD, UK Institut für Anorganische Chemie, Julius Hubland, 97074 Würzburg, Germany High Force Research Ltd., Bowburn North I 5PF, UK Department of Physics & Astronomy, Un London, WC1E 6BT, UK † Electronic supplementary informa 10.1039/c9sc00199a Cite this: Chem. Sci., 2019, 10, 4673

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 polarized 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.

In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the “hidden” degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

The bioenergetics and molecular determinants of themetabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.