Biology subjects

Kitamoto, T.

Publications and source records attributed to Kitamoto, T..

13 recordsLinked to original sources

bioRxiv2026

Drosophila glutathione S-transferase S1 exacerbates phenotypes of the gain-of-function voltage-gated sodium channel mutant paraShu through a hemocyte-mediated mechanism

Drosophila glutathione S-transferase S1 (GstS1) is a unique member of the glutathione S-transferase family and is considered the ortholog of vertebrate hematopoietic prostaglandin D synthase based on amino acid sequence similarity. A previous forward genetic screen revealed that a null mutation in GstS1 dominantly suppresses genetically induced behavioral hyperexcitability in several seizure-prone Drosophila mutants, including paraShu, a gain-of-function mutant of the voltage-gated sodium channel gene paralytic (para), and eas2, a loss-of-function mutant of the ethanolamine kinase gene easily shocked (eas). Here, using the GAL4/UAS system combined with GstS1 RNAi, we show that hemocyte-specific GstS1 knockdown suppresses hyperexcitable phenotypes in both paraShu and eas2. Notably, suppression of adult paraShu phenotypes required GstS1 knockdown before adult eclosion, suggesting that GstS1 modulates adult neuronal excitability by influencing neural development through neuroimmune interactions. Consistent with this model, hemocyte-specific GstS1 knockdown rescued abnormal dendritic morphology in developing class IV dendritic arborization (C4da) neurons of paraShu larvae. Moreover, paraShu and eas2 larvae exhibited increased numbers of sessile hemocytes surrounding C4da neuron somata, a phenotype reversed by either GstS1 knockdown or dietary supplementation with the {omega}-3 polyunsaturated fatty acid -linolenic acid. Together, these findings reveal a previously unrecognized developmental role for GstS1 in promoting nervous system hyperexcitability through hemocyte-mediated neuroimmune interactions, potentially involving bioactive lipid signaling. Our results highlight neuroimmune crosstalk as an important mechanism linking genetic perturbations to altered neural development and seizure-associated hyperexcitability.

neuroscience↗

bioRxiv2024

Confinement stress with movement restriction suppresses male courtship in Drosophila through dopamine-dependent neuroplasticity

Stress disturbs the physiological and psychological balance in animals, leading to changes in brain function. Here, we show that stress in a small space with movement restriction (SS stress) suppresses male courtship in Drosophila and that alterations in dopamine signaling induced by SS stress are responsible for the persistence of this suppression after the stress experience. We found that SS stress activates numerous dopamine neurons in the brain. Pharmacological and genetic analyses revealed that dopamine synthesis, release, and reception are essential for the persistence of SS-stress-induced courtship suppression. A specific subset of dopamine neurons projects to the mushroom body (MB), a brain region where various sensory inputs are integrated. We identified that SS-stress-induced dopamine release plastically depresses the activity of a subset of MB neurons, and this neuronal depression contributes to the sustained suppression of male courtship behavior following stress exposure. This novel stress model using Drosophila provides valuable insights into dopamine-mediated stress mechanisms, particularly those related to confined spaces.

neuroscience↗

bioRxiv2023

Calorie Restriction activates a gastric Notch-FOXO1 pathway to expand Ghrelin cells

Calorie restriction increases lifespan. While some tissue-specific protective effects of calorie restriction have been described, the impact of calorie restriction on the gastrointestinal tract remains unclear. We found increased abundance of chromogranin A+, including orexigenic ghrelin+, endocrine cells in the stomach of calorie-restricted mice. This effect coincided with increased Notch target Hes1 and Notch ligand Jag1 and was reversed when Notch signaling was blocked using the {gamma}-secretase inhibitor DAPT. Using primary cultures and genetically-modified reporter mice, we determined that increased endocrine cell abundance was due to altered stem and progenitor proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, calorie restriction triggered nuclear localization of FOXO1, which was sufficient to promote endocrine cell differentiation. Taken together, the data indicate that calorie restriction promotes gastric endocrine cell differentiation triggered by active Notch signaling and regulated by FOXO1.

cell biology↗

bioRxiv2022

Single-agent Foxo1 inhibition normalizes glycemia and induces gut β-like cells in streptozotocin-diabetic mice

Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive {beta}-like cells by targeted inhibition of Foxo1. We have shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. In the present study, we provide evidence that two novel Foxo1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut {beta}-like cell-inducing properties in mice rendered insulin-deficient by administration of streptozotocin. FBT432 is also highly effective in combination with a Notch inhibitor in this model. The data add to a growing body of evidence suggesting that Foxo1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

pharmacology and toxicology↗

bioRxiv2022

Cyb5r3-based mechanism and reversal of secondary failure to sulfonylurea

Sulfonylureas (SU) are effective and affordable anti-diabetic drugs. But chronic use leads to secondary failure, limiting their utilization. The mechanism of secondary failure is unknown. Here we identify Cyb5r3 downregulation as a mechanism of SU failure and successfully reverse it. Chronic exposure to SU impairs Cyb5r3 levels and reduces islet glucose utilization with a metabolomics signature characterized by low acetyl-CoA and amino acid levels. Cyb5r3 engages in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Accordingly, activating Gck mutations in patients with hyperinsulinemia reduce Cyb5r3 binding, whereas inactivating MODY mutations increase it, providing evidence for a role of Cyb5r3 in determining flux through Gck. The Cyb5r3 activator tetrahydroindenoindole (THII) rescues secondary failure to SU in an animal model of chronic SU treatment and restores insulin secretion from ex vivo islets. We conclude that Cyb5r3 loss-of-function is a key factor in the secondary failure to SU and a potential target for its prevention, which may lead to a rehabilitation of SU use in diabetes.

physiology↗

bioRxiv2022

Triple Notch/Tgfβ/FoxO1 blockade converts multiple intestinal sub-lineages into β-like cells and lowers glycemia in diabetic animals

Insulin is the essential treatment of Type 1 (T1D) and is often used in Type 2 Diabetes. For nearly five decades, efforts have been focused on replenishing {beta}-cells in T1D patients as a more durable treatment. Gut endocrine cells can be converted into insulin-producing cells, but their numbers are limited. In this study we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine, in addition to enteroendocrine cells. Accordingly, lineage tracing experiments show that, besides enterochromaffin cells, the Paneth/goblet lineage can undergo conversion to the insulin lineage upon genetic or pharmacologic Foxo1 ablation in mice. We leveraged these data to design a screening platform in organoids to accurately quantitate {beta}-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process by expanding the intestinal secretory lineage. We identified a triple blockade of FoxO1, Notch, and Tgf{beta} that, when tested in insulin-deficient diabetic animals resulted in a near-normalization of glucose levels, associated with the appearance of gut insulin-producing cells. The findings illustrate a therapeutic approach to replace insulin treatment in diabetes.

cell biology↗

bioRxiv2021

Chemical induction of gut β-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes

Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive {beta}-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate {beta}-like gut cells. Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced {beta}-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by small molecule FoxO1 inhibitor, Cpd10. Cpd10 induced {beta}-like cells that released insulin in response to glucose in mouse gut organoids, and this effect was strengthened by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced insulin-immunoreactive {beta}-like cells in the gut, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. These results provide proof of principle of gut cell conversion into {beta}-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications. One Sentence SummaryOrally available small molecule FoxO1 inhibitor phenocopied genetic FoxO1 ablation in generating gut {beta}-like cells

developmental biology↗

bioRxiv2020

Consolidation and maintenance of Drosophila long-term memory require LIM homeodomain protein Apterous in distinct brain neurons

The LIM-homeodomain (LIM-HD) transcription factor Apterous (Ap) and its cofactor Chip (Chi) form a complex that regulates various developmental events in Drosophila. Although Ap continues to be expressed in the adult brain, the functions of the centrally expressed Ap remain incompletely understood. Here, we show that Ap and Chi in the Drosophila memory center, the mushroom bodies (MBs), are indispensable for long-term memory (LTM) maintenance, whereas Ap in a subset of clock neurons [large ventral-lateral neurons (l-LNvs)] plays a crucial role in memory consolidation in a Chi-independent manner. Ex vivo imaging revealed that Ap, but not Chi, in l-LNvs is essential for the appropriate Cl- responses to GABA. Furthermore, knockdown of GABAA receptor in l-LNvs compensated for the impairment of memory consolidation in ap null mutant flies. Our results indicate that Drosophila Ap functions differently in l-LNvs and MBs, and it contributes to the consolidation and maintenance of LTM.

neuroscience↗

bioRxiv2020

Reduced function of the glutathione S-transferase S1 suppresses behavioral hyperexcitability in Drosophila expressing a mutant voltage-gated sodium channel

Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu. Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss. Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

genetics↗

bioRxiv2019

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure

ObjectiveDiabetes is characterized by pancreatic {beta}-cell dedifferentiation. Dedifferentiating {beta}-cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the {beta}-cells response to an adverse metabolic environment with impaired mitochondrial function remains unclear.\n\nMethodsHere we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to {beta}-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and NAD/NADH ratios.\n\nResultsExpression of Cyb5r3 is decreased in FoxO1-deficient {beta}-cells. Mice with {beta}-cell-specific deletion of Cyb5r3 have impaired insulin secretion resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient {beta}-cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient {beta}-cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability.\n\nConclusionsThe findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate {beta}-cell failure.

molecular biology↗

bioRxiv2019

Environmental light is required for maintenance of long-term memory in Drosophila

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship-conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide Pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light-dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.\n\nSignificant StatementTemporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite its critical importance, the molecular and cellular underpinnings of LTM maintenance remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling.

neuroscience↗

bioRxiv2018

Age of onset in genetic prion disease and the design of preventive clinical trials

Regulatory agencies worldwide have adopted programs to facilitate drug development for diseases where the traditional approach of a randomized trial with a clinical endpoint is expected to be prohibitively lengthy or difficult. Here we provide quantitative evidence that this criterion is met for the prevention of genetic prion disease. We assemble age of onset or death data from N=1,094 individuals with high penetrance mutations in the prion protein gene (PRNP), generate survival and hazard curves, and estimate statistical power for clinical trials. We show that, due to dramatic and unexplained variability in age of onset, randomized preventive trials would require hundreds or thousands of at-risk individuals in order to be statistically powered for an endpoint of clinical onset, posing prohibitive cost and delay and likely exceeding the number of individuals available for such trials. Instead, the characterization of biomarkers suitable to serve as surrogate endpoints will be essential for the prevention of genetic prion disease. Biomarker-based trials may require post-marketing studies to confirm clinical benefit. Parameters such as longer trial duration, increased enrollment, and the use of historical controls in a post-marketing study could provide opportunities for subsequent determination of clinical benefit.

neuroscience↗

bioRxiv2017

Evolutionarily Conserved Roles For Blood-Brain Barrier Xenobiotic Transporters In Endogenous Steroid Partitioning And Behavior

Optimal brain function depends upon efficient control over the brain entry of blood components; this is provided by the blood-brain barrier (BBB). Curiously, some brain-impermeable drugs can still cause behavioral side effects.\n\nTo investigate this phenomenon, we asked whether the promiscuous drug efflux transporter Mdr1 has dual functions in transporting drugs and endogenous molecules. If this is true, brain-impermeable drugs may cause behavioral side effects by affecting brain levels of endogenous molecules.\n\nUsing computational, genetic and pharmacologic approaches across diverse organisms we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids, and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that Mdr1-interacting drugs are associated with anxiety-related behaviors in humans.\n\nWe propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates, and implicates the BBB in behavioral regulation.\n\nAbbreviations

neuroscience↗