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Life Sciences May 2024Substance use disorder (SUD) affects over 48 million Americans aged 12 and over. Thus, identifying novel chemicals contributing to SUD will be critical for developing...
Substance use disorder (SUD) affects over 48 million Americans aged 12 and over. Thus, identifying novel chemicals contributing to SUD will be critical for developing efficient prevention and mitigation strategies. Considering the complexity of the actions and effects of these substances on human behavior, a high-throughput platform using a living organism is ideal. We developed a quick and easy screening assay using Caenorhabditis elegans. C. elegans prefers high-quality food (Escherichia coli HB101) over low-quality food (Bacillus megaterium), with a food preference index of approximately 0.2, defined as the difference in the number of worms at E. coli HB101 and B. megaterium over the total worm number. The food preference index was significantly increased by loperamide, a μ-opioid receptor (MOPR) agonist, and decreased by naloxone, a MOPR antagonist. These changes depended on npr-17, a C. elegans homolog of opioid receptors. In addition, the food preference index was significantly increased by arachidonyl-2'-chloroethylamide, a cannabinoid 1 receptor (CB1R) agonist, and decreased by rimonabant, a CB1R inverse agonist. These changes depended on npr-19, a homolog of CB1R. These results suggest that the conserved opioid and endocannabinoid systems modulate the food preference behaviors of C. elegans. Finally, the humanoid C. elegans strains where npr-17 was replaced with human MOPR and where npr-19 was replaced with human CB1R phenocopied the changes in food preference by the drug treatment. Together, the current results show that this method can be used to rapidly screen the potential effectors of MOPR and CB1R to yield results highly translatable to humans.
Topics: Animals; Humans; Caenorhabditis elegans; Food Preferences; Escherichia coli; Drug Inverse Agonism; Substance-Related Disorders; Analgesics, Opioid
PubMed: 38514005
DOI: 10.1016/j.lfs.2024.122580 -
PloS One 2024Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for...
Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3β expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.
Topics: Animals; Clathrin; Chromatin; Chromatin Assembly and Disassembly; Synaptic Transmission; Drosophila; Endocytosis; DNA Helicases
PubMed: 38512854
DOI: 10.1371/journal.pone.0300255 -
The Journal of Cell Biology Jun 2024Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a...
Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adaptor MADD, potentially preventing the formation of the RAB3A-MADD-KIF1A/1Bβ complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.
Topics: Humans; Axonal Transport; Axons; Homeostasis; Kinesins; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Parkinson Disease; Phosphorylation; Synaptic Vesicles; rab3A GTP-Binding Protein
PubMed: 38512027
DOI: 10.1083/jcb.202307092 -
Science Advances Mar 2024In animal models, deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is...
In animal models, deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is haploinsufficiency. Previous studies in mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and mouse embryos at gastrulation and early cardiac crescent stages. embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, was found to be overexpressed in all germ layers, and many gene expression changes observed in embryos could be attributed to overexpression. These findings establish a link between deficiency, overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in animals and Cornelia de Lange syndrome.
Topics: Animals; Mice; Cell Cycle Proteins; De Lange Syndrome; Gastrulation; Gene Expression; Mutation; Phenotype
PubMed: 38507484
DOI: 10.1126/sciadv.adl4239 -
PLoS Genetics Mar 2024Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide...
Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.
Topics: Animals; Mice; Actins; Cochlea; Formins; Genome-Wide Association Study; Hearing; Hearing Loss, High-Frequency; Mice, Knockout; Polymerization
PubMed: 38498576
DOI: 10.1371/journal.pgen.1011211 -
Fluids and Barriers of the CNS Mar 2024A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by...
BACKGROUND
A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs.
METHODS
To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish.
RESULTS
We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature.
CONCLUSIONS
These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.
Topics: Adult; Animals; Humans; ATP Binding Cassette Transporter, Subfamily G, Member 2; Blood-Brain Barrier; Endothelial Cells; HEK293 Cells; Mammals; Neoplasm Proteins; Zebrafish
PubMed: 38491505
DOI: 10.1186/s12987-024-00529-5 -
The Journal of Clinical Investigation Jan 2024Breast cancer stem cells (BCSCs) mitigate oxidative stress to maintain their viability and plasticity. However, the regulatory mechanism of oxidative stress in BCSCs...
Breast cancer stem cells (BCSCs) mitigate oxidative stress to maintain their viability and plasticity. However, the regulatory mechanism of oxidative stress in BCSCs remains unclear. We recently found that the histone reader ZMYND8 was upregulated in BCSCs. Here, we showed that ZMYND8 reduced ROS and iron to inhibit ferroptosis in aldehyde dehydrogenase-high (ALDHhi) BCSCs, leading to BCSC expansion and tumor initiation in mice. The underlying mechanism involved a two-fold posttranslational regulation of nuclear factor erythroid 2-related factor 2 (NRF2). ZMYND8 increased stability of NRF2 protein through KEAP1 silencing. On the other hand, ZMYND8 interacted with and recruited NRF2 to the promoters of antioxidant genes to enhance gene transcription in mammospheres. NRF2 phenocopied ZMYND8 to enhance BCSC stemness and tumor initiation by inhibiting ROS and ferroptosis. Loss of NRF2 counteracted ZMYND8's effects on antioxidant genes and ROS in mammospheres. Interestingly, ZMYND8 expression was directly controlled by NRF2 in mammospheres. Collectively, these findings uncover a positive feedback loop that amplifies the antioxidant defense mechanism sustaining BCSC survival and stemness.
Topics: Animals; Mice; Antioxidants; Ferroptosis; Kelch-Like ECH-Associated Protein 1; Neoplasms; Neoplastic Stem Cells; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Trans-Activators; Breast Neoplasms
PubMed: 38488001
DOI: 10.1172/JCI171166 -
The Canadian Journal of Cardiology May 2024In this article some of the recent advances in the use of noninvasive imaging applied to patients with hypertrophic cardiomyopathy (HCM) are discussed. Echocardiography... (Review)
Review
In this article some of the recent advances in the use of noninvasive imaging applied to patients with hypertrophic cardiomyopathy (HCM) are discussed. Echocardiography and cardiac computed tomography are briefly discussed with respect to their power to detect apical aneurysmal disease. Echocardiographic phenotype-genotype correlations and the use of echocardiography to characterize myocardial work are reviewed. Positron emission tomography is reviewed in the context of ischemia imaging and also in the context of the use of a new tracer that might allow for recognition of early activation of the fibrosis pathway. Next, the technical capabilities of cardiovascular magnetic resonance to measure myocardial perfusion, oxygenation, and disarray are discussed as they apply to HCM. The application of radiomics to improve prediction of sudden cardiac death is touched upon. Finally, a deep learning approach to the recognition of HCM vs phenocopies is presented as a potential future diagnostic aid in the not-too-distant future.
Topics: Humans; Cardiomyopathy, Hypertrophic; Echocardiography; Magnetic Resonance Imaging, Cine; Positron-Emission Tomography
PubMed: 38467329
DOI: 10.1016/j.cjca.2024.02.030 -
BioRxiv : the Preprint Server For... Feb 2024Classifying systemic inflammatory disorders as autoinflammatory or autoimmune provides insight into disease pathogenesis and whether treatment should target innate...
Classifying systemic inflammatory disorders as autoinflammatory or autoimmune provides insight into disease pathogenesis and whether treatment should target innate molecules and their signaling pathways or the adaptive immune response. COPA syndrome is a monogenic disorder of immune dysregulation that leads to interstitial lung disease and high-titer autoantibodies. Studies show constitutive activation of the innate immune molecule STING is centrally involved in disease. However, the mechanisms by which STING results in loss of T cell tolerance and autoimmunity in COPA syndrome or more common autoimmune diseases is not understood. Using mice, we uncovered a functional role for STING in the thymus. Single cell data of human thymus demonstrates STING is highly expressed in medullary thymic epithelial cells (mTECs) involved in processing and presenting self-antigens to thymocytes. In mice, activated STING in mTECs triggered interferon signaling, impaired macroautophagy and caused a defect in negative selection of T cells. Wild-type mice given a systemic STING agonist phenocopied the selection defect and showed enhanced thymic escape of a T cell clone targeting a self-antigen also expressed in melanoma. Our work demonstrates STING activation in TECs shapes the T cell repertoire and contributes to autoimmunity, findings important for settings that activate thymic STING.
PubMed: 38464209
DOI: 10.1101/2024.02.17.580803 -
BioRxiv : the Preprint Server For... Feb 2024The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity...
The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing hundreds of oncoproteins and disturbing the stoichiometry of protein complexes. Most inhibitors target the key component heat-shock protein 90 (HSP90). However, although classical HSP90 inhibitors are highly tumor-selective, they fail in phase 3 clinical oncology trials. These failures are at least partly due to an interference with a negative feedback loop by HSP90 inhibition, known as heat-shock response (HSR): in response to HSP90 inhibition there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock factor 1 (HSF1). We recently identified that wildtype p53 (p53) actively reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here we test the hypothesis that in HSP90-based therapies simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. Indeed, we find that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids and patient-derived organoids (PDOs). Mechanistically, upon combination therapy human CRC cells strongly upregulate p53-associated pathways, apoptosis, and inflammatory immune pathways. Likewise, in the chemical AOM/DSS CRC model in mice, dual HSF1-HSP90 inhibition strongly represses tumor growth and remodels immune cell composition, yet displays only minor toxicities in mice and normal mucosa-derived organoids. Importantly, inhibition of the cyclin dependent kinases 4 and 6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Even more important, in p53-deficient (mutp53-harboring) CRC cells, an HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR system and reduces cancer growth. Likewise, p53-mutated PDOs strongly respond to dual HSF1-HSP90 pathway inhibition and thus, providing a strategy to target CRC independent of the p53 status. In sum, activating p53 (in p53-proficient cancer cells) or inhibiting CDK4/6 (independent of the p53 status) provide new options to improve the clinical outcome of HSP90-based therapies and to enhance colorectal cancer therapy.
PubMed: 38464125
DOI: 10.1101/2024.02.22.581507