Biology subjects

Kaplan, J.

Publications and source records attributed to Kaplan, J..

14 recordsLinked to original sources

bioRxiv2025

Induction of Moderate DNA Damage Enhances Megakaryopoiesis and Platelet Production

A common side effect of poly-ADP ribose polymerase (PARP) inhibitors is low platelet counts, or thrombocytopenia, presumably mediated through platelet progenitors, megakaryocytes (MKs). MKs are large, hematopoietic cells with a polyploid, multi-lobulated nucleus. While DNA replication in MKs (endomitosis) is well studied, limited investigations have examined the impact of DNA damage and repair inhibition on megakaryopoiesis. To explore PARP inhibitor-induced thrombocytopenia, we treated mice with PARP inhibitors (niraparib and olaparib), which are approved for the treatment of solid tumors. While high-dose niraparib treatment led to thrombocytopenia, consistent with clinical observations, treatment at a lower dosage led to a significant, >1.5-fold increase in both the number of bone marrow MKs and circulating platelets. This increase was accompanied by elevated DNA damage in both MKs and MK progenitors, as measured by both {gamma}H2AX accumulation and comet assays of MKs. Notably, platelets from niraparib-treated mice were functionally normal in their response to ADP, TRAP, and collagen. Gamma-irradiation treatment similarly increased MK and platelet counts in mice, suggesting that moderate DNA damage enhances megakaryopoiesis and increases platelet counts. These data reveal a previously unknown relationship between MKs and DNA damage and present a novel target for triggering enhanced platelet production in vivo. Key PointsO_LITreatment of mice with low dose PARP inhibitors or gamma-irradiation enhances platelet counts. C_LIO_LILow dose PARP inhibitor treatment leads to increased DNA damage in MKs and MK progenitors and enhances bone marrow megakaryopoiesis. C_LI

cell biology↗

bioRxiv2024

High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension

Pathological high shear stress (HSS, 100 dyn/cm2) is generated in distal pulmonary arteries (PA) (100-500 m) in congenital heart defects and in progressive PA hypertension (PAH) with inward remodeling and luminal narrowing. Human PA endothelial cells (PAEC) were subjected to HSS versus physiologic laminar shear stress (LSS, 15 dyn/cm2). Endothelial-mesenchymal transition (EndMT), a feature of PAH not previously attributed to HSS, was observed. H3K27ac peaks containing motifs for an ETS-family transcription factor (ERG) were reduced, as was ERG-Kruppel-like factors (KLF)2/4 interaction and ERG expression. Reducing ERG by siRNA in PAEC during LSS caused EndMT; transfection of ERG in PAEC under HSS prevented EndMT. An aorto-caval shunt was preformed in mice to induce HSS and progressive PAH. Elevated PA pressure, EndMT and vascular remodeling were reduced by an adeno-associated vector that selectively replenished ERG in PAEC. Agents maintaining ERG in PAEC should overcome the adverse effect of HSS on progressive PAH.

cell biology↗

bioRxiv2023

Modeling the critical MCOR-causing deletion in mouse unveils aberrant Sox21 expression in developing and adult iris and ciliary body, and implicates Tgfb2 in MCOR-associated glaucoma and myopia.

Congenital microcoria (MCOR) is a rare hereditary developmental defect of the iris dilator muscle, frequently associated with high axial myopia and high intraocular pressure (IOP) glaucoma. The condition is caused by submicroscopic rearrangements of chromosome 13q32.1. However, the mechanisms underlying the failure of iris development and the origin of associated features remain elusive. Here, we present a 3D architecture model of the 13q32.1 region, demonstrating that MCOR-related deletions consistently disrupt the boundary between two Topologically Associating Domains (TADs). Deleting the critical MCOR-causing region in mice reveals ectopic Sox21 expression precisely aligning with Dct, each located in one of the two neighbor TADs. This observation is consistent with the TADs boundary alteration and adoption of Dct regulatory elements by the Sox21 promoter. Additionally, we identify Tgfb2 as a target gene of SOX21 and show TGFB2 accumulation in the aqueous humor of a MCOR-affected subject. Accumulation of TGFB2 is recognized for its role in glaucoma and potential impact on axial myopia. Our results highlight the importance of SOX21-TGFB2 signaling in iris development and control of eye growth and IOP. Insights from MCOR studies may provide therapeutic avenues for this condition but also for glaucoma and high myopia conditions, affecting millions of people.

genetics↗

bioRxiv2023

Pharmacological cAMP stimulation via prostaglandin receptors rescues ciliary defects in CEP290-deficient patient and mouse models

The retinas sensitivity to light depends on the primary cilium of photoreceptors, known as the outer segment (OS). OS defects are a primary cause of inherited retinal dystrophies (IRDs) and can also indicate wider ciliary dysfunctions. One such IRD is Leber congenital amaurosis type 10 (LCA10), which occurs as a monosymptomatic retinal disease or the presenting symptom of syndromic ciliopathies within the Senior-Loken-Joubert-Meckel spectrum. LCA10 patients are born blind but retain dormant photoreceptors for decades, offering the potential for reactivation. AAV-based gene therapies for IRDs cannot accommodate the large CEP290 gene, prompting the search for innovative treatments. LCA10 is caused by mutations in CEP290, which plays a vital role in ciliation, similar to cAMP signaling. Utilizing fibroblasts displaying variable CEP290 mutations and associated ciliary defects, we show that exposure to Taprenepag, a specific PGE2 receptor agonist, substantially stimulated cAMP synthesis and consistently improved cilia formation and elongation. We also found that intraperitoneal injection of Taprenepag in mice reached the retina and slowed retinal degeneration, promoting OS formation, and improving light sensitivity in a Cep290 mutant mouse. These findings demonstrate the potential of Taprenepag to treat the CEP290-related visual dysfunction and suggest evaluation for other CEP290-related organ issues and ciliopathies.

genetics↗

bioRxiv2023

Mon1a and FCHO2 are required for maintenance of Golgi architecture

Mon1a has been shown to function in the endolysosomal pathway functioning in the Mon1-Ccz1 complex and it also acts in the secretory pathway where it interacts with dynein and affects ER to Golgi traffic. Here we show that Mon1a is also required for maintenance of the Golgi apparatus. We identified the F-BAR protein FCHO2 as a Mon1a-interacting protein by both yeast two-hybrid analysis and co-immunoprecipitation. siRNA-dependent reductions in Mon1a or FCHO2 resulted in Golgi fragmentation. Membrane trafficking through the secretory apparatus in FCHO2-depleted cells was unaltered, however, reduction of FCHO2 affected the uniform distribution of Golgi enzymes necessary for carbohydrate modification. Fluorescence recovery after photobleaching analysis showed that the Golgi ministacks in Mon1a- or FCHO2-silenced cells did not exchange resident membrane proteins. The effect of FCHO2 silencing on Golgi structure was partially cell cycle-dependent and required mitosis-dependent Golgi fragmentation, whereas the effect of Mon1a-silencing on Golgi disruption was not cell cycle-dependent. mCherry-FCHO2 transiently colocalized on Golgi structures independent of Mon1a. These findings suggest that Mon1a has functions throughout the secretory pathway including interacting with dynein at the ER-Golgi interface in vesicle formation and then interacting with FCHO2 at the Golgi to generate lateral links between ministacks, thus creating Golgi ribbons.

cell biology↗

bioRxiv2022

Zn2+ potentiation of OTOP proton channels identifies structural elements of the gating apparatus

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they may mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vesibular system and it forms the receptor for sour taste, while the functions of OTOP2, and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well-understood, and until recently, it was not even known if the channels were gated. Here we show that Zn2+, as well as other transition metals including copper (Cu2+), potently activate murine OTOP3. Zn2+ pre-exposure increases the magnitude of OTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, OTOP2 currents are insensitive to potentiation by Zn2+. Swapping the extracellular tm 11-12 linker between OTOP3 and OTOP2 was sufficient to eliminate Zn2+ potentiation of OTOP3 and confer Zn2+ potentiation on OTOP2. We also show that H531 within the tm 11-12 linker is essential for potentiation of OTOP3 by Zn2+, likely by forming part of its binding site. Kinetic modeling of the data is consistent with Zn2+ stabilizing the open state of the channel, possibly competing with H+ for activation of the channels. These results establish the tm 11-12 linker as part of the gating apparatus of OTOP channels and a target for drug discovery. Zinc is an essential micronutrient and its regulation of OTOP channels will undoubtedly have important physiological sequelae. Significance StatementA family of proton-activated H+ ion channels was recently identified that includes the sour receptor OTOP1. Here we show that members of the OTOP channel family are differentially sensitive to Zn2+, which strongly activates OTOP3 but not OTOP2. By studying chimeric channels, we identify structural elements in the extracellular linker between transmembrane domains 11-12 that are necessary and sufficient for Zn2+ activation of OTOP channels. In addition to the taste system, OTOP channels play important roles in biomineralization in both vertebrate and invertebrates and are expressed in the digestive tract where their expression is a predictor of cancer prognosis. Our identification of the tm 11-12 linker as part of the gating apparatus makes it a promising target for pharmaceutical discovery.

biophysics↗

bioRxiv2022

Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

Otopetrin (OTOP) channels are proton-selective ion channels conserved among vertebrates and invertebrates and with no structural similarity to other ion channels. There are three vertebrate OTOP channels (OTOP1, OTOP2, and OTOP3), of which one (OTOP1), functions as a sour taste receptor. Whether OTOP channels are gated by, as well as permeating, protons was not known. Here, by comparing functional properties of the three vertebrate proton channels with patch-clamp recording and cytosolic pH microfluorimetry, we provide evidence that each is gated by external protons. OTOP1 and OTOP3 are both activated by extracellular protons, with a sharp threshold of pHe <6.0 and 5.5 respectively, while OTOP2 is negatively gated by protons, and more conductive at alkaline extracellular pH (>pH 9.0). Strikingly, we found that we could change pH-sensitive gating of OTOP2 and OTOP3 channels by swapping extracellular linkers that connect transmembrane domains. Swaps of linkers within the N domain changed the relative conductance at alkaline pH, while swaps within the C domain tended to change the rates of OTOP3 current activation. We conclude that members of the OTOP channel family are proton-gated (acid-sensitive) proton channels and that the gating apparatus is distributed across multiple extracellular regions within both the N and C domains of the channels. In addition to the taste system, OTOP channels are found in the vestibular and digestive systems, where pH sensitivity may be tuned to specific functions.

biophysics↗

bioRxiv2021

Male pheromones modulate synaptic transmission at the C. elegans neuromuscular junction in a sexually dimorphic manner

The development of functional synapses in the nervous system is important for animal physiology and behaviors. The synaptic transmission efficacy can be modulated by the environment to accommodate external changes, which is crucial for animal reproduction and survival. However, the underlying plasticity of synaptic transmission remains poorly understood. Here we show that in C. elegans, the male pheromone increases the hermaphrodite cholinergic transmission at the neuromuscular junction (NMJ), which alters hermaphrodites locomotion velocity and mating efficiency in a developmental stage-dependent manner. Dissection of the sensory circuits reveals that the AWB chemosensory neurons sense those male pheromones and further transduce the information to NMJ using cGMP signaling. Exposure of hermaphrodites to male pheromones specifically increases the accumulation of presynaptic CaV2 calcium channels and clustering of postsynaptic receptors at cholinergic synapses of NMJ, which potentiates cholinergic synaptic transmission. Thus, our study demonstrates a circuit mechanism for synaptic modulation by sexual dimorphic pheromones.

neuroscience↗

bioRxiv2021

Fear in the mind's eye: Mental imagery can generate and regulate acquired differential fear conditioned reactivity

Mental imagery is an important tool in the cognitive control of emotion. The present study tests the prediction that visual imagery can generate and regulate differential fear conditioning via the activation and prioritization of stimulus representations in early visual cortices. We combined differential fear conditioning with manipulations of viewing and imagining basic visual stimuli in humans. We discovered that mental imagery of a fear-conditioned stimulus compared to imagery of a safe conditioned stimulus generated a significantly greater conditioned response as measured by self-reported fear, the skin conductance response, and right anterior insula activity (experiment 1). Moreover, mental imagery effectively down- and up-regulated the fear conditioned responses (experiment 2). Multivariate classification using the functional magnetic resonance imaging data from retinotopically defined early visual regions revealed significant decoding of the imagined stimuli in V2 and V3 (experiment 1) but significantly reduced decoding in these regions during imagery-based regulation (experiment 2). Together, the present findings indicate that mental imagery can generate and regulate a differential fear conditioned response via mechanisms of the depictive theory of imagery and the biased-competition theory of attention. These findings also highlight the potential importance of mental imagery in the manifestation and treatment of psychological illnesses.

neuroscience↗

bioRxiv2021

Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

Neutrophil elastase (NE) is implicated in pulmonary arterial hypertension (PAH) but the role of neutrophils in the pathogenesis of PAH is unclear. Here we show that neutrophils from PAH vs. control subjects produce and release increased NE associated with enhanced extracellular trap formation. PAH neutrophils are highly adherent and show decreased migration consistent with increased vinculin, identified on proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic interferon signature in PAH neutrophils and an increase in human endogenous retrovirus (HERV-K) envelope protein. NE and interferon genes are induced by HERV-K envelope and vinculin is increased by HERV-K dUTPase that is elevated in PAH plasma. Neutrophil exosomes from PAH plasma contain increased NE and HERV-K envelope and induce pulmonary hypertension in mice, that is mitigated by the NE inhibitor and antiviral agent, elafin. Thus, elevated HERVs explain pathological neutrophils linked to PAH induction and progression.

immunology↗

bioRxiv2020

Breast cancer endocrine therapy exhausts adipocyte progenitors promoting weight gain and glucose intolerance

Breast cancer survivors treated with anti-estrogen therapies report weight gain and have an elevated risk of type 2 diabetes. Here, we show that current tamoxifen use associated with larger breast adipocyte diameter only in women with a BMI >30 kg/m2. To understand the mechanisms behind these clinical findings, we investigated the impact of estrogen deprivation and tamoxifen in a relevant pre-clinical model of obesity. Specifically, mature female mice were housed at thermoneutrality and fed either a low-fat/low-sucrose (LFLS) or a high-fat/high-sucrose (HFHS) diet. Consistent with the high expression of Esr1 observed in single-cell RNA sequencing of mesenchymal stem cells from adipose tissue, endocrine therapies induced adipose accumulation and preadipocyte expansion, but resulted in adipocyte progenitor depletion only in the context of HFHS. Consequently, 7-week endocrine therapy supported adipocyte hypertrophy and was associated with hepatic steatosis, hyperinsulinemia, insulin resistance, and glucose intolerance, particularly in HFHS fed females. Metformin or pioglitazone, glucose lowering drugs used to treat diabetes, prevented the effects of tamoxifen but not estrogen deprivation on adipocyte size and insulin resistance in HFHS-fed mice. This translational study suggests that endocrine therapies act via ER to directly disrupt adipocyte progenitors and support adipocyte hypertrophy, leading to ectopic lipid deposition that may promote hyperinsulinemia, insulin resistance and type 2 diabetes. Interventions that target insulin action should be considered for some women receiving life-saving endocrine therapies for breast cancer.

physiology↗

bioRxiv2020

Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits

BackgroundSkeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases, mobility, and quality of life. It is composed of several different cell and muscle fiber types. ResultsHere, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell-specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 30,531 nuclei, representing 11 libraries, profiled in this study, and identify seven distinct cell types ranging in abundance from 63% (type II fibers) to 0.9% (muscle satellite cells) of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site-distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells ([~]3% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, and transcription factor motifs for creatinine levels and type 2 diabetes signals. ConclusionsThese chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for investigating specific cell types and nominating causal GWAS SNPs and cell types.

genomics↗

bioRxiv2020

Dopachrome tautomerase variants in patients with oculocutaneous albinism.

PurposeAlbinism is a clinically and genetically heterogeneous condition. Despite analysis of the nineteen known genes, [~]30% patients remain unsolved. We aimed to identify new genes involved in albinism. MethodsWe sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. ResultsWe identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14 bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR/Cas9 was used in C57BL/6J mice to create mutations identical to the missense mutations carried by the patients, along with one loss-of-function indel mutation. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared to Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanocytes and RPE cells. ConclusionsDCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8.

genetics↗

bioRxiv2019

Mechanistic characterization of RASGRP1 variants identifies an hnRNP K-regulated transcriptional enhancer contributing to SLE susceptibility

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component. We recently identified a novel SLE susceptibility locus near RASGRP1, which governs the ERK/MAPK kinase cascade and B-/T-cell differentiation and development. However, precise causal RASGRP1 functional variant(s) and their mechanisms of action in SLE pathogenesis remain undefined. Our goal was to fine-map this locus, prioritize genetic variants according to likely functionality, experimentally validate the contribution of three SNPs to SLE risk, and experimentally determine their biochemical mechanisms of action. We performed a meta-analysis across six Asian and European cohorts (9,529 cases; 22,462 controls), followed by in silico bioinformatic and epigenetic analyses to prioritize potentially functional SNPs. We experimentally validated the functional significance and mechanism of action of three SNPs in cultured T-cells. Meta-analysis identified 18 genome-wide significant (p<5x10-8) SNPs, mostly concentrated in two haplotype blocks, one intronic and the other intergenic. Epigenetic fine-mapping, allelic, eQTL and imbalance analyses predicted three transcriptional regulatory regions with four SNPs (rs7170151, rs11631591-rs7173565, and rs9920715) prioritized for functional validation. Luciferase reporter assays indicated significant allele-specific enhancer activity for intronic rs7170151 and rs11631591-rs7173565 in T-lymphoid (Jurkat) cells, but not in HEK293 cells. Following up with EMSA, mass spectrometry and ChIP-qPCR, we detected allele-dependent interactions between heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and rs11631591. Furthermore, inhibition of hnRNP-K in Jurkat and primary T-cells downregulated RASGRP1 and ERK/MAPK signaling. Comprehensive association, bioinformatics, and epigenetic analyses yielded putative functional variants of RASGRP1, which were experimentally validated. Notably, intronic variant (rs11631591) is located in a cell type-specific enhancer sequence, where its risk allele binds to the hnRNP-K protein and modulates RASGRP1 expression in Jurkat and primary T-cells. As risk allele dosage of rs11631591 correlates with increased RASGRP1 expression and ERK activity, we suggest that this SNP may underlie SLE risk at this locus.

genetics↗