Biology subjects

Schlichting, M.

Publications and source records attributed to Schlichting, M..

12 recordsLinked to original sources

bioRxiv2024

Chronic mitochondrial fragmentation elicits a neuroprotective Warburg-like effect in Drosophila neurons

Mitochondrial fission and fusion are dynamic and important cellular processes, but the roles of these two very different mitochondrial forms - predominantly spherical and tubular - are not well-characterized in neurons of animals and especially in aging neurons. This is important because neurons are long-lived and mitochondrial dynamics is associated with neurodegenerative diseases. We used here an efficient cell type-specific CRISPR approach to knockout key fission-fusion genes and disrupt mitochondrial dynamics within the inessential clock neurons of Drosophila. Surprisingly, fusion is much more important than fission for maintaining long-term neuronal function. Neurons survive chronic mitochondrial fragmentation due to loss of fusion by triggering a cancer-like transcriptomic response. This Warburg effect includes ATF4-mediated upregulation of the aerobic glycolysis gene Lactate dehydrogenase (Ldh), and LDH is essential to prevent neurodegeneration of neurons deficient in the fusion gene Opa1. These results and others provide insights into the intersection of neuronal metabolism, aging and neurodegeneration.

neuroscience↗

bioRxiv2024

Metabolic control of adaptive β-cell proliferation by the protein deacetylase SIRT2

Selective and controlled expansion of endogenous {beta}-cells has been pursued as a potential therapy for diabetes. Ideally, such therapies would preserve feedback control of {beta}-cell proliferation to avoid excessive {beta}-cell expansion and an increased risk of hypoglycemia. Here, we identified a regulator of {beta}-cell proliferation whose inactivation results in controlled {beta}-cell expansion: the protein deacetylase Sirtuin 2 (SIRT2). Sirt2 deletion in {beta}-cells of mice increased {beta}-cell proliferation during hyperglycemia with little effect in homeostatic conditions, indicating preservation of feedback control of {beta}-cell mass. SIRT2 restrains proliferation of human islet {beta}-cells cultured in glucose concentrations above the glycemic set point, demonstrating conserved SIRT2 function. Analysis of acetylated proteins in islets treated with a SIRT2 inhibitor revealed that SIRT2 deacetylates enzymes involved in oxidative phosphorylation, dampening the adaptive increase in oxygen consumption during hyperglycemia. At the transcriptomic level, Sirt2 inactivation has context-dependent effects on {beta}-cells, with Sirt2 controlling how {beta}-cells interpret hyperglycemia as a stress. Finally, we provide proof-of-principle that systemic administration of a GLP1-coupled Sirt2-targeting antisense oligonucleotide achieves {beta}-cell selective Sirt2 inactivation and stimulates {beta}-cell proliferation under hyperglycemic conditions. Overall, these studies identify a therapeutic strategy for increasing {beta}-cell mass in diabetes without circumventing feedback control of {beta}-cell proliferation.

physiology↗

bioRxiv2023

Development of human hippocampal subfield microstructure and relation to associative inference

The hippocampus is a complex brain structure composed of subfields that each have distinct cellular organizations. While the volume of hippocampal subfields displays age-related changes that have been associated with inference and memory functions, the degree to which the cellular organization within each subfield is related to these functions throughout development is not well understood. We employed an explicit model testing approach to characterize the development of tissue microstructure and its relationship to performance on two inference tasks, one that required memory (memory-based inference) and one that required only perceptually available information (perception-based inference). We found that each subfield had a unique relationship with age in terms of its cellular organization. While the subiculum (SUB) displayed a linear relationship with age, the dentate gyrus (DG), cornu ammonis field 1 (CA1), and cornu ammonis subfields 2 and 3 (combined; CA2/3) displayed non-linear trajectories that interacted with sex in CA2/3. We found that the DG was related to memory-based inference performance and that the SUB was related to perception-based inference; neither relationship interacted with age. Results are consistent with the idea that cellular organization within hippocampal subfields might undergo distinct developmental trajectories that support inference and memory performance throughout development.

neuroscience↗

bioRxiv2022

Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-{beta}-cells, a hallmark of {beta}-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-{beta}-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-{beta}-cells. Correspondingly, BMP4 augmented the SC-{beta}-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between {beta}-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

cell biology↗

bioRxiv2021

Dopamine and GPCR-mediated modulation of DN1 clock neurons gates the circadian timing of sleep

The metronome-like circadian regulation of sleep timing must still adapt to an uncertain environment. Recent studies in Drosophila indicate that neuromodulation not only plays a key role in clock neuron synchronization but also affects interactions between the clock network and brain sleep centers. We show here that the targets of neuromodulators, G-Protein Coupled Receptors (GPCRs), are highly enriched in the fly brain circadian clock network. Single cell sequencing indicates that they are not only differentially expressed but also define clock neuron identity. We generated a comprehensive guide library to mutagenize individual GPCRs in specific neurons and verified the strategy with a targeted sequencing approach. Combined with a behavioral screen, the mutagenesis strategy revealed a novel role of dopamine in sleep regulation by identifying two dopamine receptors and a clock neuron subpopulation that gate the timing of sleep.

neuroscience↗

bioRxiv2021

Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase

Many species synchronize their physiology and behavior to specific hours. It is commonly assumed that sunlight acts as the main entrainment signal for ~24h clocks. However, the moon provides similarly regular time information, and increasingly studies report correlations between diel behavior and lunidian cycles. Yet, mechanistic insight into the possible influences of the moon on ~24hr timers is scarce. We studied Platynereis dumerilii and uncover that the moon, besides its role in monthly timing, also schedules the exact hour of nocturnal swarming onset to the nights darkest times. Moonlight adjusts a plastic clock, exhibiting <24h (moonlit) or >24h (no moon) periodicity. Abundance, light sensitivity, and genetic requirement indicate Platynereis r-Opsin1 as receptor to determine moonrise, while the cryptochrome L-Cry is required to discriminate between moon- and sunlight valence. Comparative experiments in Drosophila suggest that Cryptochromes requirement for light valence interpretation is conserved. Its exact biochemical properties differ, however, between species with dissimilar timing ecology. Our work advances the molecular understanding of lunar impact on fundamental rhythmic processes, including those of marine mass spawners endangered by anthropogenic change.

neuroscience↗

bioRxiv2020

A transcriptomic taxonomy of Drosophila circadian neurons around the clock

Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila circadian neurons, we optimized a single cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. We suggest that these molecules orchestrate the temporal regulation of synapse formation and/or strength. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.

neuroscience↗

bioRxiv2020

Internal state configures olfactory behavior and early sensory processing in Drosophila larva

Animals exhibit different behavioral responses to the same sensory cue depending on their state at a given moment in time. How and where in the brain are sensory inputs combined with internal state information to select an appropriate behavior? Here we investigate how food deprivation affects olfactory behavior in Drosophila larvae. We find that certain odors reliably repel well-fed animals but attract food-deprived animals. We show that feeding state flexibly alters neural processing in the first olfactory center, the antennal lobe. Food deprivation differentially modulates two separate output pathways that are required for opposing behavioral responses. Uniglomerular projection neurons mediate odor attraction and show elevated odor-evoked activity in the food-deprived state. A multiglomerular projection neuron mediates odor aversion and receives odor-evoked inhibition in the food-deprived state. The switch between these two pathways is regulated by the lone serotonergic neuron in the antennal lobe, CSD. Our findings demonstrate how flexible behaviors can arise from state-dependent circuit dynamics in an early sensory processing center.

neuroscience↗

bioRxiv2019

Single cell chromatin accessibility reveals pancreatic islet cell type- and state-specific regulatory programs of diabetes risk

Genetic risk variants for complex, multifactorial diseases are enriched in cis-regulatory elements. Single cell epigenomic technologies create new opportunities to dissect cell type-specific mechanisms of risk variants, yet this approach has not been widely applied to disease-relevant tissues. Given the central role of pancreatic islets in type 2 diabetes (T2D) pathophysiology, we generated accessible chromatin profiles from 14.2k islet cells and identified 13 cell clusters including multiple alpha, beta and delta cell clusters which represented hormone-producing and signal-responsive cell states. We cataloged 244,236 islet cell type accessible chromatin sites and identified transcription factors (TFs) underlying both lineage- and state-specific regulation. We measured the enrichment of T2D and glycemic trait GWAS for the accessible chromatin profiles of single cells, which revealed heterogeneity in the effects of beta cell states and TFs on fasting glucose and T2D risk. We further used machine learning to predict the cell type-specific regulatory function of genetic variants, and single cell co-accessibility to link distal sites to putative cell type-specific target genes. We localized 239 fine-mapped T2D risk signals to islet accessible chromatin, and further prioritized variants at these signals with predicted regulatory function and co-accessibility with target genes. At the KCNQ1 locus, the causal T2D variant rs231361 had predicted effects on an enhancer with beta cell-specific, long-range co-accessibility to the insulin promoter, and deletion of this enhancer reduced insulin gene and protein expression in human embryonic stem cell-derived beta cells. Our findings provide a cell type- and state-resolved map of gene regulation in human islets, illuminate likely mechanisms of T2D risk at hundreds of loci, and demonstrate the power of single cell epigenomics for interpreting complex disease genetics.

genomics↗

bioRxiv2019

Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.

neuroscience↗

bioRxiv2019

Light-mediated circuit switching in the Drosophila neuronal clock network

The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas Cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. Here we show that a single photoreceptor sub-type is essential for long day adaptation. These cells activate key circadian neurons, namely the lLNvs, which release the neuropeptide PDF. Using a cell-specific CRISPR/Cas9 assay, we show that PDF directly interacts with neurons important for evening (E) activity timing. Interestingly, this pathway is specific for light entrainment and appears to be dispensable in constant darkness (DD). The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which is a novel form of plasticity.

neuroscience↗

bioRxiv2018

Allatostatin C/AstC-R2 is a Novel Pathway to Modulate Circadian Activity Pattern in Drosophila

Six neuropeptides are expressed within the Drosophila brain circadian network. Our previous mRNA profiling suggested that AllatostatinC is a seventh neuropeptide and specifically expressed in dorsal clock neurons (DN1s). Our results here show that AstC is indeed expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral posterior neurons (LPNs). Behavioral experiments indicate that clock neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd neuron. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the behavioral response to changing photoperiod in Drosophila.

neuroscience↗