Biology subjects

Taylor, M. R.

Publications and source records attributed to Taylor, M. R..

9 recordsLinked to original sources

bioRxiv2025

Cytoplasmic CaMKIIδ-B prevents myocardial recovery in heart failure

Restoration of cardiac function in patients with advanced heart failure is rare, and the molecular processes that regulate recovery are unknown. To identify potential mechanisms, we studied paired myocardial samples before and after left ventricular assist device therapy, where significant cardiac functional recovery occurred in [~]25% of patients. We found that expression of the nuclear B isoform of Ca2+/calmodulin-dependent protein kinase II{delta} (CaMKII{delta}-B) inversely correlated with recovery. Furthermore, increased phosphorylation near the CaMKII{delta}-B nuclear localization signal in non-responders prevented its auto-activation dependent nuclear translocation. Expression of a cytoplasm-restricted CaMKII{delta}-B in cardiomyocytes dramatically remodeled the phospho-proteome and impaired contractility, while a nuclear-competent version did not. Modulating CaMKII{delta} subcellular localization may thus represent a therapeutic strategy for advanced heart failure.

physiology↗

bioRxiv2023

IL-1β disrupts blood-brain barrier development by inhibiting endothelial Wnt/β-catenin signaling

During neuroinflammation, the proinflammatory cytokine Interleukin-1{beta} (IL-1{beta}) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1{beta} on the in vivo development of the BBB. We generated a doxycycline-inducible transgenic zebrafish model that drives secretion of Il-1{beta} in the CNS. To validate the utility of our model, we showed Il-1{beta} dose-dependent mortality, recruitment of neutrophils, and expansion of microglia. Using live imaging, we discovered that Il-1{beta} causes a significant reduction in CNS angiogenesis and barriergenesis. To demonstrate specificity, we rescued the Il-1{beta} induced phenotypes by targeting the zebrafish il1r1 gene using CRISPR/Cas9. Mechanistically, we determined that Il-1{beta} disrupts BBB development by decreasing Wnt/{beta}-catenin transcriptional activation in brain endothelial cells. Given that several neurodevelopmental disorders are associated with inflammation, our findings support further investigation into the connections between proinflammatory cytokines, neuroinflammation, and neurovascular development.

cell biology↗

bioRxiv2023

The N-terminal region of DNMT3A combines multiple chromatin reading motifs to guide recruitment.

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns. However, the mechanisms underlying DNMT3A recruitment to and function within different chromatin environments remain unclear. Using a combination of biochemical and structural approaches we find that DNMT3A interacts using multiple interfaces with chromatin; directly binding generic nucleosome features as well as site-specific post-translational histone modifications. The N-terminal region, unique to the DNMT3A1 isoform, is essential for these interactions and stabilises H3K36me2-nucleosome recruitment. Intriguingly, in the same region critical for nucleosome binding we also map a ubiquitylation-dependent recruitment motif (UDR). The UDR binds specifically to ubiquitylated H2AK119, explaining the previously observed recruitment to Polycomb-occupied heterochromatin. A cryo-EM structure of DNMT3A1-DNMT3L with a modified nucleosome reveals that the UDR interacts with the nucleosome surface including the acidic patch. Previously unexplained disease-associated mutations are present in the UDR and ablate nucleosome interactions. This leads to an increased understanding of how DNMT3A1 recruitment occurs in the genome and highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome.

biochemistry↗

bioRxiv2023

Modular Splicing is Linked to Evolution in the Synapse-Specificity Molecule Kirrel3

Kirrel3 is a cell-adhesion molecule that instructs the formation of specific synapses during brain development in mouse and Kirrel3 variants may be risk factors for autism and intellectual disabilities in humans. Kirrel3 is predicted to undergo alternative splicing but brain isoforms have not been studied. Here, we present the first in-depth characterization of Kirrel3 isoform diversity in brain using targeted, long-read mRNA sequencing of mouse hippocampus. We identified 19 isoforms with predicted transmembrane and secreted forms and show that even rare isoforms generate detectable protein in the brain. We also analyzed publicly-available long-read mRNA databases from human brain tissue and found 11 Kirrel3 isoforms that, similar to mouse, encode transmembrane and secreted forms. In mice and humans, Kirrel3 diversity arises from alternative, independent use of protein-domain coding exons and alternative early translation-stop signals. Intriguingly, the alternatively spliced exons appear at branch points in the chordate phylogenetic tree, including one exon only found in humans and their closest living relatives, the great apes. Together, these results validate a simple pipeline for analyzing isoform diversity in genes with low expression and suggest that Kirrel3 function is fine-tuned by alternative splicing and may play a role in brain evolution. Significance StatementKirrel3 is an important molecule for synapse and circuit formation with gene variants that are associated with neurodevelopmental disorders, yet Kirrel3 function remains largely unknown. Here, we report new isoforms of mouse and human Kirrel3, including secreted and transmembrane forms, that suggest a diverse repertoire of Kirrel3 actions. Importantly, we identified a new Kirrel3 exon only present in humans and the other great apes with potential to play an important role in circuit formation unique to these species.

neuroscience↗

bioRxiv2023

Innate Immune Signaling in Hearts and Buccal Mucosa Cells of Patients with Arrhythmogenic Cardiomyopathy

STRUCTURED ABSTRACTO_ST_ABSObjectivesC_ST_ABSWe sought to determine if persistent innate immune signaling via NF{kappa}B occurs in cardiac myocytes in patients with arrhythmogenic cardiomyopathy and if this is associated with myocardial infiltration of pro-inflammatory cells expressing CCR2. We also determined if buccal mucosa cells from young subjects with inherited disease alleles exhibit NF{kappa}B signaling. BackgroundNF{kappa}B signaling in cardiac myocytes causes disease in a mouse model of arrhythmogenic cardiomyopathy by mobilizing CCR2-expressing macrophages which promote myocardial injury and arrhythmias. Buccal mucosa cells exhibit pathologic features similar to those seen in cardiac myocytes in patients with arrhythmogenic cardiomyopathy. MethodsWe analyzed myocardium from arrhythmogenic cardiomyopathy patients who died suddenly or required cardiac transplantation. We also analyzed buccal mucosa cells from young subjects with inherited disease alleles. The presence of immunoreactive signal for RelA/p65 in nuclei of cardiac myocytes and buccal cells was used as a reliable indicator of active NF{kappa}B signaling. We also counted myocardial CCR2-expressing cells. ResultsNF{kappa}B signaling was seen in cardiac myocytes in 34 of 36 cases of arrhythmogenic cardiomyopathy but in none of 19 age-matched controls. Cells expressing CCR2 were increased in patient hearts in numbers directly correlated with the number of cardiac myocytes showing NF{kappa}B signaling. NF{kappa}B signaling also occurred in buccal cells in young subjects with active disease. ConclusionsPatients with clinically active arrhythmogenic cardiomyopathy exhibit persistent innate immune responses in cardiac myocytes and buccal mucosa cells reflecting an inflammatory process that fails to resolve. Such individuals may benefit from anti-inflammatory therapy. CONDENSED ABSTRACTNF{kappa}B signaling in cardiac myocytes causes arrhythmias and myocardial injury in a mouse model of arrhythmogenic cardiomyopathy by mobilizing pro-inflammatory CCR2-expressing macrophages to the heart. Based on these new mechanistic insights, we analyzed hearts of arrhythmogenic cardiomyopathy patients who died suddenly or required cardiac transplantation. We observed active NF{kappa}B signaling in cardiac myocytes associated with marked infiltration of CCR2-expressing cells. We also observed NF{kappa}B signaling in buccal mucosa cells obtained from young subjects with active disease. Thus, anti-inflammatory therapy may be effective in arrhythmogenic cardiomyopathy. Screening buccal cells may be a reliable way to identify patients most likely to benefit. HIGHLIGHTS- Inflammation likely contributes to the pathogenesis of arrhythmogenic cardiomyopathy but the responsible mechanisms and the roles of specific classes of immune cells remain undefined. - NF{kappa}B signaling in cardiac myocytes is sufficient to cause disease in a mouse model of arrhythmogenic cardiomyopathy by mobilizing injurious myeloid cells expressing CCR2 to the heart. - Here, we provide evidence of persistent NF{kappa}B signaling in cardiac myocytes and increased CCR2-expressing cells in hearts of patients with arrhythmogenic cardiomyopathy. We observed a close correlation between the number of cardiac myocytes with active NF{kappa}B signaling and the number of CCR2-expressing cells in patient hearts. - We also provide evidence of active NF{kappa}B signaling in buccal mucosa cells associated with initial onset of disease and/or disease progression in young subjects with arrhythmogenic cardiomyopathy alleles.

pathology↗

bioRxiv2023

Histone divergence in Trypanosoma brucei results in unique alterations to nucleosome structure

Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically and structurally characterised nucleosome core particles (NCPs) from the kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened overall DNA binding. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.

biochemistry↗

bioRxiv2019

Kirrel3-mediated synapse formation is attenuated by disease-associated missense variants

Missense variants in Kirrel3 are repeatedly identified as risk factors for autism spectrum disorder and intellectual disability but it has not been reported if or how these variants disrupt Kirrel3 function. Previously, we studied Kirrel3 loss-of-function using knockout mice and showed that Kirrel3 is a synaptic adhesion molecule necessary to form one specific type of hippocampal synapse in vivo. Here, we developed a new gain-of-function assay for Kirrel3 and find that wild-type Kirrel3 induces synapse formation selectively between Kirrel3-expressing cells via homophilic, trans-cellular binding. We tested six disease-associated Kirrel3 missense variants and find that five attenuate this synaptogenic function. All variants tested traffic to the cell surface and localize to synapses similar to wild-type Kirrel3. Two tested variants lack homophilic trans-cellular binding, which likely accounts for their reduced synaptogenic function. Interestingly, we also identified variants that bind in trans but cannot induce synapses, indicating Kirrel3 trans-cellular binding is necessary but not sufficient for its synaptogenic function. Collectively, these results suggest Kirrel3 functions as a synaptogenic, cell-recognition molecule, and this function is attenuated by missense variants associated with autism spectrum disorder and intellectual disability. Thus, we provide critical insight to Kirrel3 function in typical brain development and the consequences of missense variants associated with autism spectrum disorder and intellectual disability. SIGNIFICANCE STATEMENTHere, we advance our understanding of mechanisms mediating target-specific synapse formation by providing evidence that Kirrel3 trans-cellular interactions mediate contact recognition and signaling to promote synapse development. Moreover, this is the first study to test the effects of disease-associated Kirrel3 missense variants on synapse formation, and thereby, provides a framework to understand the etiology of complex neurodevelopmental disorders arising from rare missense variants in synaptic genes.

neuroscience↗

bioRxiv2019

Arl13b regulates Sonic Hedgehog signaling from outside primary cilia

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13Bs role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

developmental biology↗

bioRxiv2019

Vancomycin Relieves Mycophenolate Mofetil-Induced Gastrointestinal Toxicity by Eliminating Gut Bacterial β-Glucuronidase Activity

Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has proven advantages over other immunosuppressive drugs but gastrointestinal (GI) side effects frequently limit its use. The pathways involved in the metabolism of the prodrug MMF and the bioactive derivative mycophenolic acid (MPA) are well characterized but the mechanism responsible for toxicity is unknown. Here we extend our previous observation that an intact gut microbiome is required for MMF-induced toxicity and demonstrate that gut bacterial metabolism is responsible for the GI inflammation and weight loss associated with MMF exposure. In mice consuming MMF, the introduction of vancomycin alone was sufficient to prevent or reverse MMF-induced weight loss and colonic inflammation. MMF induced the expansion of bacteria expressing {beta}-glucuronidase (GUS) in the cecum and proximal colon. GUS activity, which is responsible for the catabolism of glucuronidated MPA (MPAG) to free MPA, was increased in the presence of MMF and eliminated by vancomycin. Vancomycin eliminated multiple Bacteroides spp. that flourished in the presence of MMF and prevented the breakdown of MPAG without negatively affecting serum MPA levels. Human data suggests that increased stool GUS activity can be associated with MMF-related toxicity. Our work provides a mechanism for the GI toxicity associated with MMF and a future direction for the development of therapeutics.

microbiology↗