Biology subjects

Knott, M.

Publications and source records attributed to Knott, M..

4 recordsLinked to original sources

bioRxiv2023

Potentiating NaV1.1 in Dravet syndrome patient iPSC-derived GABAergic neurons increases neuronal firing frequency and decreases network synchrony

Dravet syndrome is a developmental and epileptic encephalopathy characterized by seizures, behavioral abnormalities, developmental deficits, and elevated risk of sudden unexpected death in epilepsy (SUDEP). Most patient cases are caused by de novo loss-of-function mutations in the gene SCN1A, causing a haploinsufficiency of the alpha subunit of the voltage-gated sodium channel NaV1.1. Within the brain, NaV1.1 is primarily localized to the axons of inhibitory neurons, and decreased NaV1.1 function is hypothesized to reduce GABAergic inhibitory neurotransmission within the brain, driving neuronal network hyperexcitability and subsequent pathology. We have developed a human in vitro model of Dravet syndrome using differentiated neurons derived from patient iPSC and enriched for GABA expressing neurons. Neurons were plated on high definition multielectrode arrays (HD-MEAs), permitting recordings from the same cultures over the 7-weeks duration of study at the network, single cell, and subcellular resolution. Using this capability, we characterized the features of axonal morphology and physiology. Neurons developed increased spiking activity and synchronous network bursting. Recordings were processed through a spike sorting pipeline for curation of single unit activity and to assess the effects of pharmacological treatments. At 7-weeks, the application of the GABAAR receptor agonist muscimol eliminated network bursting, indicating the presence of GABAergic neurotransmission. To identify the role of NaV1.1 on neuronal and network activity, cultures were treated with a dose-response of the NaV1.1 potentiator {delta}-theraphotoxin-Hm1a. This resulted in a strong increase in firing rates of putative GABAergic neurons, an increase in the intraburst firing rate, and eliminated network bursting. These results validate that potentiation of NaV1.1 in Dravet patient iPSC-derived neurons results in decreased firing synchrony in neuronal networks through increased GABAergic neuron activity and support the use of human neurons and HD-MEAs as viable high-throughput electrophysiological platform to enable therapeutic discovery.

neuroscience↗

bioRxiv2022

A novel lineage of RORγt+Aire+ antigen presenting cells promotes peripheral generation of intestinal regulatory T cells and tolerance during early life

Establishing and maintaining tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new class of ROR{gamma}t+ antigen-presenting cells (APC), dubbed Thetis cells (TCs), with transcriptional features of both mTECs and dendritic cells (DCs), comprising 4 major sub-groups (TC I-IV). We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While TC I and III expressed the signature mTEC nuclear factor Aire, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-{beta} activating integrin v{beta}8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by ROR{gamma}t+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the tolerogenic ROR{gamma}t+ APC with an essential early life function. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.

immunology↗

bioRxiv2021

Visual Clustering of Transcriptomic Data from Primary and Metastatic Tumors: Dependencies and Novel Pitfalls

Personalized Oncology is a rapidly evolving area and offers cancer patients therapy options more specific than ever. Yet, there is still a lack of understanding regarding transcriptomic similarities or differences of metastases and corresponding primary sites. Applying two unsupervised dimension reduction methods (t-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation Projection (UMAP)) on three datasets of metastases (n=682 samples) with three different data transformations (unprocessed, log10 as well as log10+1 transformed values), we visualized potential underlying clusters. Additionally, we analyzed two datasets (n=616 samples) containing metastases and primary tumors of one entity, to point out potential familiarities. Using these methods, no tight link between site of resection and cluster formation outcome could be demonstrated, neither for datasets consisting of solely metastasis nor mixed datasets. Instead, dimension reduction methods and data transformation significantly impacted visual clustering results. Our findings strongly suggest data transformation to be considered as another key element in interpretation of visual clustering approaches along with initialization and different parameters. Furthermore, the results indicate only minor transcriptional differences for metastases and corresponding primary tumors.

cancer biology↗

bioRxiv2018

Cooperation of dominant oncogenes with regulatory germline variants shapes clinical outcomes in childhood cancer

INTRODUCTORY PARAGRAPHDeciphering principles of inter-individual tumor heterogeneity is essential for refinement of personalized anti-cancer therapy. Unlike cancers of adulthood, pediatric malignancies including Ewing sarcoma (EwS) feature a striking paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome.\n\nHere we demonstrate in the EwS model how cooperation of a dominant oncogene and regulatory variants determine tumor growth, patient survival and drug response.\n\nWe show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2, whose high expression promotes poor patient outcome via activation of pro-proliferative signatures. Analysis of paired germline and tumor whole-genome sequencing data revealed that regulatory variability at this locus is inherited via the germline. CRISPR-mediated interference with this regulatory element almost abolished MYBL2 transcription, and MYBL2 knockdown decreased cell proliferation, cell survival and tumorigenicity of EwS cells. Combined RNA- and ChIP-seq analyses as well as functional experiments and clinical data identified CCNF, BIRC5 and AURKB as direct MYBL2 targets and critical mediators of its phenotype. In drug-response experiments, high MYBL2 levels sensitized EwS cells for inhibition of its activating cyclin dependent kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a predictive biomarker for targeted anti-CDK2-therapy.\n\nCollectively, our findings establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and indicate the importance of integrating the regulatory genome in the process of developing new diagnostic and/or therapeutic strategies to fully harness the potential of precision medicine.

cancer biology↗