Biology subjects

Jones-Tabah, J.

Publications and source records attributed to Jones-Tabah, J..

8 recordsLinked to original sources

bioRxiv2025

N-terminal acetylation reduces α-synuclein pathology in models of Parkinson's disease

The -synuclein protein, encoded by SNCA gene, is a major constituent of pathological intracellular inclusions such as Lewy bodies found in the brains of patients with Parkinsons disease and other synucleinopathies. Whereas -synuclein phosphorylation has been much studied, comparatively less work has been devoted to other post-translational modifications such as acetylation, especially given that N-terminally acetylated -synuclein is the most abundant endogenous form of the protein in the brain. In this study, using multiple in vitro and in vivo models, we sought to better understand the role of N-terminal acetylation in the pathogenesis of synucleinopathies. We found that N-terminal acetylation slowed aggregation of both -synuclein monomers and pre-formed fibrils in vitro. Uptake of acetylated -synuclein pre-formed fibrils into both immortalized cell lines and iPSC-derived dopamine neurons was also slowed compared non-acetylated fibrils. In addition, exposure to acetylated pre-formed fibrils induced less seeding of endogenous -synuclein, as measured by the accumulation of Serine129-phosphorylated -synuclein inclusions in both iPSC-derived dopamine neurons and mouse brain. Finally, mice injected with N-terminally acetylated -synuclein pre-formed fibrils survived significantly longer than mice injected with non-acetylated fibrils. Taken together, our study indicates that N-terminal acetylation reduces -synuclein aggregation, uptake into cells, seeding of endogenous -synuclein, and toxicity in vivo, suggesting that this prevalent post-translational modification represents a potent, physiologically relevant protective mechanism, which has thus far largely not been taken into consideration in most experimental paradigms of Parkinsons disease and synucleinopathies.

molecular biology↗

bioRxiv2023

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinsons disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

neuroscience↗

bioRxiv2022

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. Post-mortem tissue or biopsies from diseased patients, which remain scarcely available, rendered it possible to study end-stage heart disease yet the inclusion of healthy human cardiac materials for basic science research was beyond reach. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human inducible stem cell derived cardiomyocytes presented a renewed opportunity. We think it necessary to conduct a comparative study, assessing cellular signalling through cardiac G protein-coupled receptors and bulk transcriptomics of traditional rat neonatal cardiomyocytes and human iPSC-CMs. Genetically-encoded biosensors were used to interrogate nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) in rat and human-derived cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted for an in-depth examination of existing differences between both in vitro cardiomyocyte models. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human CMs. In line with this, bulk RNA-seq demonstrated key differences regarding the expression patterns of GPCRs, G proteins and effectors. Overall, our study demonstrates that human stem cell derived models of the cardiomyocyte do provide significant advantages and should be taken advantage of.

biochemistry↗

bioRxiv2022

Effective use of genetically-encoded optical biosensors for profiling signalling signatures in iPSC-CMs derived from idiopathic dilated cardiomyopathy patients

Dilated cardiomyopathy (DCM) is a cardiovascular condition that develops when the left ventricle of the heart enlarges, compromising its function and diminishing its capacity to pump oxygenated blood throughout the body. After patients are diagnosed with DCM, disease progression can lead to heart failure and the need for a heart transplantation. DCM is a complex disease where underlying causes can be idiopathic, genetic, or environmental. An incomplete molecular understanding of disease progression poses challenges for drug discovery efforts as effective therapeutics strategies remain elusive. Decades of research using primary cells or animal models have increased our understanding of DCM but has been hampered due to the inaccessibility of human cardiomyocytes, to model cardiac disease, in vitro, in a dish. Here, our goal is to leverage patient-derived hiPSC-CMs and to combine them with biosensors to understand how cellular signalling is altered in DCM. With high sensitivity and versatility, optical biosensors represent the ideal tools to dissect the molecular determinants of cardiovascular disease, in an unbiased manner and in real-time at the level of single cells. By characterizing the pathobiology of dilated cardiomyopathy in a patient-specific manner using high content biosensor-based assays, we aim to uncover personalized mechanisms for the occurrence and development of DCM and as a pathway to development of personalized therapeutics.

biochemistry↗

bioRxiv2021

Dopamine D1 receptor activation and cAMP/PKA signalling mediate Brd4 recruitment to chromatin to regulate gene expression in rat striatal neurons

The activity of striatal medium-spiny projection neurons is regulated by dopamine acting principally at D1 and D2 dopamine receptors. The dopamine D1 receptor (D1R) is a Gs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Transcriptional regulation downstream of the D1R involves the activation of PKA, which can translocate to the nucleus to phosphorylate various targets. The chromatin reader Brd4 regulates transcription induced by neurotrophic factors in cortical neurons and has also been implicated in dopamine-dependent striatal functions. Brd4 is activated by phosphorylation; this facilitates its binding to acetylated histones at promoters and enhancers. In non-neuronal cells, Brd4 is recruited to chromatin in response to PKA signalling. However, it is unknown whether Brd4 is involved in transcriptional activation by the D1R in striatal neurons. Here, we demonstrate that cAMP/PKA signalling increases Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown or inhibition of Brd4 modulated D1R-induced gene expression. Specifically, inhibition of Brd4 with the bromodomain inhibitor JQ1 suppressed the expression of [~]25% of D1R-upregulated genes, while increasing the expression of a subset of immediate-early genes, including Fos and Jun. This pro-transcriptional effect of JQ1 was P-TEFb-dependent, and mediated through inhibition of the BD1 bromodomain of Brd4. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify Brd4 as a novel regulator of D1R-dependent transcription and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

neuroscience↗

bioRxiv2021

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional "bulk" analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

neuroscience↗

bioRxiv2020

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Like many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell-type specific, and can change in response to disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinsons disease (PD), alterations in this pathway lead to activation of extracellular regulated kinases (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Forster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

neuroscience↗

bioRxiv2018

A novel interaction between Gβγ and RNA polymerase II regulates cardiac fibrosis.

G{beta}{gamma} subunits are involved in many different signalling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear G{beta}{gamma}, we investigated the functional role of G{beta}{gamma} signalling in regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. Following activation of the angiotensin II type I receptor in these cells, G{beta}{gamma} dimers interact with RNA polymerase II (RNAPII). Our findings suggest that G{beta}1 recruitment to RNAPII negatively regulates the fibrotic transcriptional response, which can be overcome by strong fibrotic stimuli. The interaction between G{beta}{gamma} subunits and RNAPII expands the role for G{beta}{gamma} signalling in cardiac fibrosis. The G{beta}{gamma}-RNAPII interaction was regulated by signaling pathways in HEK 293 cells that diverged from those operating in cardiac fibroblasts. Thus, the interaction may be a conserved feature of transcriptional regulation although such regulation may be cell-specific.

biochemistry↗