Biology subjects

Keeley, S.

Publications and source records attributed to Keeley, S..

5 recordsLinked to original sources

bioRxiv2026

High levels of the cardiomyocyte-specific kinase Tnni3k impair zebrafish heart regeneration by driving chronic myocardial inflammation

Background: Zebrafish regenerate their hearts after injury, and defining the barriers that block this capacity in mammals may reveal targets for heart failure treatment. Elevated levels of the cardiomyocyte-specific kinase TNNI3K are associated with human cardiomyopathy, and its overexpression drives adverse remodeling in mice. Recent work has linked elevated TNNI3K to cardiomyocyte polyploidization and loss of regenerative competence, but whether this cell-cycle effect accounts for the pathology seen in patients has not been established. Because TNNI3K is restricted to cardiomyocytes, whether it also acts non-cell-autonomously is unknown. Methods: We generated an allelic series of zebrafish lines comprising cardiomyocyte-specific Tnni3k overexpression, an expression-matched kinase-dead variant, a full-locus deletion, and a line overexpressing mouse Tnni3k. Regeneration, cardiomyocyte proliferation, and ploidy were assessed in uninjured hearts and after cryoinjury. We combined ventricular RNA-sequencing, macrophage depletion before or after injury, tamoxifen-inducible Cre-lox switches, and cardiomyocyte-restricted CRISPR mutagenesis. Hearts from mice overexpressing human TNNI3K were analyzed in parallel. Results: Elevated Tnni3k impaired heart regeneration in a kinase-dependent manner. Cardiomyocyte proliferation, redifferentiation, and productive cell division were preserved, and polyploidization remained well below the threshold compatible with robust regeneration, arguing against a cell-cycle defect. Instead, Tnni3k overexpression established an inflammatory and fibrotic state in the uninjured myocardium, marked by interferon, NF-{kappa}B, and antigen-presentation programs and by leukocyte and macrophage accumulation. This state amplified after injury, and these animals retained substantially more scar at 60 days post-injury. Depleting macrophages before injury, but not after, prevented the excess fibrosis. Switching Tnni3k off after injury attenuated both inflammation and fibrosis. Cardiomyocyte-specific deletion of pkmb, a glycolytic gene strongly repressed in Tnni3k-overexpressing animals and previously associated with inflammation, reproduced both phenotypes. Mice overexpressing TNNI3K showed comparable fibrosis and macrophage accumulation. Conclusions: Elevated Tnni3k impairs cardiac regeneration by sustaining a chronic inflammatory and fibrotic state rather than by driving polyploidization. Because this state requires continued kinase activity and remains reversible after injury, TNNI3K inhibition may warrant exploration as a strategy to interrupt the inflammation-fibrosis loop in inflammation-driven cardiac disease.

developmental biology↗

bioRxiv2026

Tracking the Fidelity of Internal Neural Representations with Error-In-Variables Regression

Internal neural representations can systematically deviate from externally measured sensory and behavioral variables, yet neuroscientists lack a principled statistical framework to quantify these mismatches. Here we introduce a nonlinear error-in-variables regression framework that explicitly models neural activity as a function of latent internal variables that deviate from measured sensory and behavioral variables. This approach uses a flexible basis expansion and a sampling-based inference scheme to jointly infer neuron-specific tuning functions, latent trajectories, and a representational fidelity parameter{kappa} that controls the strength of coupling between latent and measured variables. On synthetic datasets, the model accurately recovers latent dynamics, tuning curves, and identifies the true fidelity regime via cross-validated marginal likelihood. Applied to population recordings from mouse anterodorsal thalamic nucleus and rat medial entorhinal cortex across distinct sensory and behavioral conditions, the framework reveals condition-dependent changes in representational fidelity, tuning gain and profile, and uncovers latent population manifolds that are obscured in conventional tuning analyses. These results establish error-in-variables regression as a powerful and computationally tractable tool for tracking the fidelity of internal neural representations in systems neuroscience experiments.

neuroscience↗

bioRxiv2025

Improved inference of latent neural states from calcium imaging data

Calcium imaging (CI) is a standard method for recording neural population activity, as it enables simultaneous recording of hundreds-to-thousands of individual somatic signals. Accordingly, CI recordings are prime candidates for population-level latent variable analyses, for example, using models such as Gaussian Process Factor Analysis (GPFA), hidden Markov models (HMMs), and latent dynamical systems. However, these models have been primarily developed and fine-tuned for electrophysiological measurements of spiking activity. To adapt these models for use with the calcium signals recorded with CI, per-neuron fluorescence time-traces are typically either de-convolved to approximate spiking events or analyzed directly under Gaussian observation assumptions. The former approach, while enabling the direct application of latent variable methods developed for spiking data, suffers from the imprecise nature of spike estimation from CI. Moreover, isolated spikes can be undetectable in the fluorescence signal, creating additional uncertainty. A more direct model linking observed fluorescence to latent variables would account for these sources of uncertainty. Here, we develop accurate and tractable models for characterizing the latent structure of neural population activity from CI data. We propose to augment HMM, GPFA, and dynamical systems models with a CI observation model that consists of latent Poisson spiking and autoregressive calcium dynamics. Importantly, this model is both more flexible and directly compatible with standard methods for fitting latent models of neural dynamics. We demonstrate that using this more accurate CI observation model improves latent variable inference and model fitting on both CI observations generated using state-of-the-art biophysical simulations and imaging data recorded in an experimental setting. We expect the developed methods to be widely applicable to many different analyses of population CI data.

neuroscience↗

bioRxiv2025

LRH-1 is a novel regulator of neutrophil-driven immune responses within the tumor microenvironment.

Elevated plasma cholesterol levels have been linked to worse outcomes in breast and ovarian cancer. Prior work including our own has demonstrated that myeloid immune cells are highly responsive to cholesterol fluctuations and to proteins involved in cholesterol regulation. However, the specific roles of Liver Receptor Homolog-1 (LRH-1, or NR5A2), a key transcriptional regulator of cholesterol homeostasis, within myeloid cells remains largely undefined. Interestingly, LRH-1 mRNA levels are reduced in both breast and ovarian tumors compared to normal tissue. Its elevated expression within tumors is associated with increased survival time. These clinical correlations prompted us to explore the role of LRH-1 in myeloid cells particularly in the context of breast and ovarian cancer progression. Initial analyses confirmed LRH-1 expression in various myeloid cell types, with particularly high levels in neutrophils. We therefore focused on how LRH-1 influences neutrophil behaviors relevant to cancer, including migration, NETosis, phagocytosis, and interactions with T cells. Small molecule ligands for LRH-1 regulated neutrophil migration towards cancer cells. Phagocytosis was also regulated by LRH-1 small molecule ligands, the extent being dependent on type of bait (e. coli vs. cancer cells). In T cell co-cultures, neutrophils pretreated with an LRH-1 agonist promoted greater T cell expansion - particularly in CD4 cells - while LRH-1 inhibition suppressed this response. Moreover, LRH-1 activation reduced NETosis, while treatment with an antagonist or inverse agonist enhanced NETosis. Treatment of mice with an inverse agonist of LRH-1 increased the growth of 4T1 and E0771 mammary tumors. Given prior evidence that neutrophils contribute to the recurrence of dormant lesions, we further examined the role of LRH-1 in this context. Mice harboring dormant D2.0R mammary cancer lesions treated with an LRH-1 inverse agonist exhibited earlier tumor recurrence and enhanced metastatic progression compared to vehicle-treated controls. In contrast, treatment with BL001, a small chemical LRH-1 agonist, delayed recurrence in D2.0R-grafted mice. Collectively, these findings reveal that LRH-1 is a novel regulator of neutrophil-driven immune responses within the tumor microenvironment. LRH-1 thus remerges as a promising therapeutic target to suppress metastasis or prevent recurrence in breast cancer.

cancer biology↗

bioRxiv2024

Optimization of methods for rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system

CRISPR/Cas9 has massively accelerated the generation of gene loss-of-function models in zebrafish. However, establishing tissue-specific mutant lines remains a laborious and time-consuming process. Although a few dozen tissue-specific Cas9 zebrafish lines have been developed, the lack of standardization of some key methods, including gRNA delivery, has limited the implementation of these approaches in the zebrafish community. To tackle these limitations, we have established a cardiomyocyte-specific Cas9 line, the cardiodeleter, which efficiently generates biallelic mutations in combination with gene-specific gRNAs. We have also optimized the development of transposon-based guide shuttles that carry gRNAs targeting a gene of interest and permanently label the cells susceptible to becoming mutant. We validated this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), all resulting in the loss of the corresponding protein or phenocopying established mutants. Additionally, we provide detailed protocols describing how to generate guide shuttles, which will facilitate the dissemination of these techniques in the zebrafish community. Our approach enables the rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, bypassing limitations such as embryonic lethality, making it a valuable tool for cell-autonomous studies and genetic screenings.

developmental biology↗