NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis.

Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.

High-content analysis of neuronal morphology in human iPSC-derived neurons.

We present a high-content analysis (HCA) protocol for monitoring the outgrowth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). We describe steps to perform HCA imaging, followed by quantifying the morphology of dendrites and axons within a high-throughput system to evaluate neurons obtained through various differentiation approaches. This protocol can be used to screen for modulators of neuronal morphogenesis or neurotoxicity. The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al. (2021). The protocol can be used in combination with Zink et al. (2022).

High-content screening of mitochondrial polarization in neural cells derived from human pluripotent stem cells.

We present a high-content screening (HCS) protocol for quantifying mitochondrial activity in live neural cells from human induced pluripotent stem cells (iPSCs). The assessment is based on mitochondrial membrane potential, which is influenced by the efficiency of mitochondrial bioenergetics. We describe how to perform the analysis using both an HCS platform and the open-source software CellProfiler. The protocol can identify the mitochondrial fitness of human neurons and may be used to carry out high-throughput compound screenings in patient-derived neural cells. For complete details on the use and execution of this protocol, please refer to Lorenz et al. (2017) and Zink et al. (2020).

Myelinating Co-Culture as a Model to Study Anti-NMDAR Neurotoxicity.

Anti-NMDA receptor (NMDAR) encephalitis is frequently associated with demyelinating disorders (e.g., multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-associated disease (MOGAD)) with regard to clinical presentation, neuropathological and cerebrospinal fluid findings. Indeed, autoantibodies (AABs) against the GluN1 (NR1) subunit of the NMDAR diminish glutamatergic transmission in both neurons and oligodendrocytes, leading to a state of NMDAR hypofunction. Considering the vital role of oligodendroglial NMDAR signaling in neuron-glia communication and, in particular, in tightly regulated trophic support to neurons, the influence of GluN1 targeting on the physiology of myelinated axon may be of importance. We applied a myelinating spinal cord cell culture model that contains all major CNS cell types, to evaluate the effects of a patient-derived GluN1-specific monoclonal antibody (SSM5) on neuronal and myelin integrity. A non-brain reactive (12D7) antibody was used as the corresponding isotype control. We show that in cultures at the late stage of myelination, prolonged treatment with SSM5, but not 12D7, leads to neuronal damage. This is characterized by neurite blebbing and fragmentation, and a reduction in the number of myelinated axons. However, this significant toxic effect of SSM5 was not observed in earlier cultures at the beginning of myelination. Anti-GluN1 AABs induce neurodegenerative changes and associated myelin loss in myelinated spinal cord cultures. These findings may point to the higher vulnerability of myelinated neurons towards interference in glutamatergic communication, and may refer to the disturbance of the NMDAR-mediated oligodendrocyte metabolic supply. Our work contributes to the understanding of the emerging association of NMDAR encephalitis with demyelinating disorders.

Modeling Drosophila gut microbe interactions reveals metabolic interconnectivity.

We know a lot about varying gut microbiome compositions. Yet, how the bacteria affect each other remains elusive. In mammals, this is largely based on the sheer complexity of the microbiome with at least hundreds of different species. Thus, model organisms such as Drosophila melanogaster are commonly used to investigate mechanistic questions as the microbiome consists of only about 10 leading bacterial species. Here, we isolated gut bacteria from laboratory-reared Drosophila, sequenced their respective genomes, and used this information to reconstruct genome-scale metabolic models. With these, we simulated growth in mono- and co-culture conditions and different media including a synthetic diet designed to grow Drosophila melanogaster. Our simulations reveal a synergistic growth of some but not all gut microbiome members, which stems on the exchange of distinct metabolites including tricarboxylic acid cycle intermediates. Culturing experiments confirmed our predictions. Our study thus demonstrates the possibility to predict microbiome-derived growth-promoting cross-feeding.

Bone remodelling patterns around orthodontic mini-implants migrating in bone: an experimental study in rat vertebrae.

BACKGROUND: Orthodontic implant migration has been clinically observed in presence of continuous loading forces. Recent studies indicate that osteocytes play a crucial role in this phenomenon. OBJECTIVES: Aim of this study was to investigate local osteocytic gene expression, protein expression, and bone micro-structure in peri-implant regions of pressure and tension. MATERIAL AND METHODS: The present work reports a complementary analysis to a previous micro-computed tomography study. Two customized mini-implants were placed in one caudal rat vertebra and connected by a nickel-titanium contraction spring generating different forces (i.e. 0, 0.5, 1.0, and 1.5 N). Either at 2 or 8 weeks, the vertebrae were harvested and utilized for 1. osteocytic gene expression using laser capture micro-dissection on frozen sections coupled with qPCR, 2. haematoxylin-eosin staining for qualitative and quantitative analyses, 3. immunofluorescence staining and analysis, and 4. bone-to-implant contact on undecalcified samples. RESULTS: At the two time points for all the performed analyses no significant differences were observed with respect to the applied force magnitudes and cell harvesting localization. However, descriptive histological analysis revealed remarkable bone remodelling at 2 weeks of loading. At 8 weeks the implants were osseointegrated and, especially in 1.0 and 1.5 N groups, newly formed bone presented a characteristic load bearing architecture with trabecula oriented in the direction of the loading. CONCLUSIONS: The present study confirmed that stress-induced bone remodelling is the biological mechanism of orthodontic implant migration. Bone apposition was found at 'tension' and 'pressure' sites thus limiting implant migration over time.

Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones.

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.

Lipid Droplet Contact Sites in Health and Disease.

After having been disregarded for a long time as inert fat drops, lipid droplets (LDs) are now recognized as ubiquitous cellular organelles with key functions in lipid biology and beyond. The identification of abundant LD contact sites, places at which LDs are physically attached to other organelles, has uncovered an unexpected level of communication between LDs and the rest of the cell. In recent years, many disease factors mutated in hereditary disorders have been recognized as LD contact site proteins. Furthermore, LD contact sites are dramatically rearranged in response to infections with intracellular pathogens, as well as under pathological metabolic conditions such as hepatic steatosis. Collectively, it is emerging that LD-organelle contacts are important players in development and progression of disease.

Visualization of endogenous gut bacteria in Drosophila melanogaster using fluorescence in situ hybridization.

All metazoans are colonized by a complex and diverse set of microorganisms. The microbes colonize all parts of the body and are especially abundant in the gastrointestinal tract, where they constitute the gut microbiome. The fruit fly Drosophila melanogaster turned out to be an exquisite model organism to functionally test the importance of an intact gut microbiome. Still, however, fundamental questions remain unanswered. For example, it is unknown whether a fine-tuned regionalization of the gut microbiome exists and how such a spatial organization could be established. In order to pave the way for answering this question, we generated an optimized and adapted fluorescence in situ hybridization (FISH) protocol. We focused on the detection of the two major Drosophila gut microbiome constituting bacteria genera: Acetobacter and Lactobacillus. FISH allows to detect the bacteria in situ and thus to investigate their spatial localization in respect to the host as well as to other microbiome members. We demonstrate the applicability of the protocol using a diverse set of sample types.

High-Content C. elegans Screen Identifies Natural Compounds Impacting Mitochondria-Lipid Homeostasis and Promoting Healthspan.

The aging process is concurrently shaped by genetic and extrinsic factors. In this work, we screened a small library of natural compounds, many of marine origin, to identify novel possible anti-aging interventions in Caenorhabditis elegans, a powerful model organism for aging studies. To this aim, we exploited a high-content microscopy platform to search for interventions able to induce phenotypes associated with mild mitochondrial stress, which is known to promote animal's health- and lifespan. Worms were initially exposed to three different concentrations of the drugs in liquid culture, in search of those affecting animal size and expression of mitochondrial stress response genes. This was followed by a validation step with nine compounds on solid media to refine compounds concentration, which led to the identification of four compounds (namely isobavachalcone, manzamine A, kahalalide F and lutein) consistently affecting development, fertility, size and lipid content of the nematodes. Treatment of Drosophila cells with the four hits confirmed their effects on mitochondria activity and lipid content. Out of these four, two were specifically chosen for analysis of age-related parameters, kahalalide F and lutein, which conferred increased resistance to heat and oxidative stress and extended animals' healthspan. We also found that, out of different mitochondrial stress response genes, only the C. elegans ortholog of the synaptic regulatory proteins neuroligins, nlg-1, was consistently induced by the two compounds and mediated lutein healthspan effects.

CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo.

The Seipin protein is a conserved key component in the biogenesis of lipid droplets (LDs). Recently, a cooperation between human Seipin and the Lipid droplet assembly factor 1 (LDAF1) was described. LDAF1 physically interacts with Seipin and the holocomplex safeguards regular LD biogenesis. The function of LDAF1 proteins outside mammals is less clear. In yeast, the lipid droplet organization (LDO) proteins, which also cooperate with Seipin, are the putative homologs of LDAF1. While certain functional aspects are shared between the LDO and mammalian LDAF1 proteins, the relationship between the proteins is under debate. Here, we identify the Drosophila melanogaster protein CG32803, which we re-named to dmLDAF1, as an insect member of this protein family. dmLDAF1 decorates LDs in cultured cells and in vivo and the protein is linked to the fly and mouse Seipin proteins. Altering the dmLDAF1 abundance affects LD size, number and overall lipid storage amounts. Our results suggest that the LDAF1 proteins thus fulfill an evolutionarily conserved function in the biogenesis and biology of LDs.

A stem cell based in vitro model of NAFLD enables the analysis of patient specific individual metabolic adaptations in response to a high fat diet and AdipoRon interference.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disease. Its development and progression depend on genetically predisposed susceptibility of the patient towards several 'hits' that induce fat storage first and later inflammation and fibrosis. Here, we differentiated induced pluripotent stem cells (iPSCs) derived from four distinct donors with varying disease stages into hepatocyte like cells (HLCs) and determined fat storage as well as metabolic adaptations after stimulations with oleic acid. We could recapitulate the complex networks that control lipid and glucose metabolism and we identified distinct gene expression profiles related to the steatosis phenotype of the donor. In an attempt to reverse the steatotic phenotype, cells were treated with the small molecule AdipoRon, a synthetic analogue of adiponectin. Although the responses varied between cells lines, they suggest a general influence of AdipoRon on metabolism, transport, immune system, cell stress and signalling.

Grease on-Perspectives in lipid droplet biology.

Lipid droplets (LDs) are now recognized as omnipresent and dynamic subcellular organelles of amazing morphological and functional diversity. Beyond the obvious benefit of having molecules full of chemical energy stored in a dedicated structural entity, LDs may also be viewed as a safe harbor for potentially damaging metabolites. This protective function might in many cases even supersede the relevance of lipid storage for eventual energy gain and membrane biogenesis. Furthermore, the LD surface constitutes a unique membrane environment, creating a platform for hosting specific proteins and thus enabling their interactions. These metabolic hotspots would contribute decisively to compartmentalized metabolism in the cytosol. LDs are also communicating extensively with other subcellular organelles in directing and regulating lipid metabolism. Deciphering the relevance of LD storage and regulation at the organismic level will be essential for the understanding of widespread and serious metabolic complications in humans. Increasing attention is also devoted to pathogens appropriating LDs for their own benefit. LD biology is still considered an emerging research area in rapid and vibrant development, attracting scientists from all disciplines of the life sciences and beyond, which is mirrored by the accompanying review collection. Here, we present our personal views on areas we believe are especially exciting and hold great potential for future developments. Particularly, we address issues relating to LD biogenesis and heterogeneity, required technological advances, and the complexity of human physiology.

FlySilico: Flux balance modeling of Drosophila larval growth and resource allocation.

Organisms depend on a highly connected and regulated network of biochemical reactions fueling life sustaining and growth promoting functions. While details of this metabolic network are well established, knowledge of the superordinate regulatory design principles is limited. Here, we investigated by iterative wet lab and modeling experiments the resource allocation process during the larval development of Drosophila melanogaster. We chose this system, as survival of the animals depends on the successful allocation of their available resources to the conflicting processes of growth and storage metabolite deposition. First, we generated "FlySilico", a curated metabolic network of Drosophila, and performed time-resolved growth and metabolite measurements with larvae raised on a holidic diet. Subsequently, we performed flux balance analysis simulations and tested the predictive power of our model by simulating the impact of diet alterations on growth and metabolism. Our predictions correctly identified the essential amino acids as growth limiting factor, and metabolic flux differences in agreement with our experimental data. Thus, we present a framework to study important questions of resource allocation in a multicellular organism including process priorization and optimality principles.

Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER.

During energy bursts, neutral lipids fabricated within the ER bilayer demix to form lipid droplets (LDs). LDs bud off mainly in the cytosol where they regulate metabolism and multiple biological processes. They indeed become accessible to most enzymes and can interact with other organelles. How such directional emergence is achieved remains elusive. Here, we found that this directionality is controlled by an asymmetry in monolayer surface coverage. Model LDs emerge on the membrane leaflet of higher coverage, which is improved by the insertion of proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate by remodeling ER shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from ER.

Control of Drosophila Growth and Survival by the Lipid Droplet-Associated Protein CG9186/Sturkopf.

Lipid droplets (LDs) are the universal cellular storage organelles for esterified neutral lipids. The increasing number of characterized LD-associated proteins attained LDs with hitherto unexpected functions on top of their classical role as energy depot. Here, we characterize the LD-associated protein CG9186 of Drosophila by a CRISPR/Cas9-derived mutant fly line. While the mutant flies only showed a mild triacylglycerol storage phenotype, they were highly protected from desiccation stress, likely linked to a reduced locomotor activity and altered cuticular hydrocarbons. Both parameters depend on juvenile hormone (JH) signaling. Together with an observed interaction between CG9186 and JH-degrading enzymes, our results suggest that CG9186 participates in endocrine physiology regulation. In support of this hypothesis, CG9186 mutant flies show an altered expression of JH target genes and fail to adjust their developmental rate to dietary yeast-to-sugar ratio changes. Our results thus link LDs to organismic physiology regulation.

The impact of genome variation and diet on the metabolic phenotype and microbiome composition of Drosophila melanogaster.

The metabolic phenotype of an organism depends on a complex regulatory network, which integrates the plethora of intrinsic and external information and prioritizes the flow of nutrients accordingly. Given the rise of metabolic disorders including obesity, a detailed understanding of this regulatory network is in urgent need. Yet, our level of understanding is far from completeness and complicated by the discovery of additional layers in metabolic regulation, such as the impact of the microbial community present in the gut on the hosts' energy storage levels. Here, we investigate the interplay between genome variation, diet and the gut microbiome in the shaping of a metabolic phenotype. For this purpose, we reared a set of fully sequenced wild type Drosophila melanogaster flies under basal and nutritionally challenged conditions and performed metabolic and microbiome profiling experiments. Our results introduce the fly as a model system to investigate the impact of genome variation on the metabolic response to diet alterations and reveal candidate single nucleotide polymorphisms associated with different metabolic traits, as well as metabolite-metabolite and metabolite-microbe correlations. Intriguingly, the dietary changes affected the microbiome composition less than anticipated. These results challenge the current view of a rapidly changing microbiome in response to environmental fluctuations.

Targeting of the Drosophila protein CG2254/Ldsdh1 to a subset of lipid droplets.

Lipid droplets (LDs) are the principal organelles of lipid storage. They consist of a hydrophobic core of storage lipids, surrounded by a phospholipid monolayer with proteins attached. While some of these proteins are known to be essential for the regulation of cellular and organismic lipid metabolism, key questions concerning LD protein function, such as their targeting to LDs, are still unanswered. Intriguingly, some proteins are restricted to subsets of LDs by an as-yet-unknown mechanism. This finding makes LD targeting even more complex. Here, we characterize the Drosophila protein CG2254, which is targeted to subsets of LDs in cultured cells and in different larval Drosophila tissues, where the prevalence of subsets of LDs appears highly dynamic. We find that an amphipathic amino acid stretch mediates CG2254 LD localization. Additionally, we identified a juxtaposed sequence stretch limiting CG2254 localization to a subset of LDs. This sequence is sufficient to restrict a chimeric protein consisting of the subset-targeting sequence introduced to an otherwise pan-LD-localized protein sequence to a subset of LDs. Based on its subcellular localization and annotated function, we suggest that CG2254 is renamed Lipid droplet subset dehydrogenase 1 (Ldsdh1).

The why, when and how of lipid droplet diversity.

Lipid droplets are the universal cellular organelles for the transient or long-term storage of lipids. The number, size and composition of lipid droplets vary greatly within cells in a homogenous population as well as in different cell types. The variability of intracellular lipid-storage organelles reflects the diversification of lipid droplet composition and function. Lipid droplet diversification results, for example, in two cellular lipid droplet populations that are prone to diminish and grow, respectively. The aberrant accumulation or depletion of lipids are hallmarks or causes of various human pathologies. Thus, a better understanding of the origins of lipid droplet diversification is not only a fascinating cell biology question but also potentially serves to improve comprehension of pathologies that entail the accumulation of lipids. This Commentary covers the lipid droplet life cycle and highlights the early steps during lipid droplet biogenesis, which we propose to be the potential driving forces of lipid droplet diversification.