Document text
Principal Investigator: Douglas A. Bayliss
Organization: UNIVERSITY OF VIRGINIA
Fiscal Year: 2024
Award: $474,809
Funding agency: National Heart Lung and Blood Institute
A discrete group of neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor,
Phox2b provide a crucial excitatory drive to regulate downstream respiratory rhythm/pattern-generating circuits. The activity of these neurons is modulated by changes in CO2 (or its proxy, H+) and various other sensory
and arousal-state inputs to control breathing; their dysfunction is implicated in various central disorders of
breathing (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). The molecular and
cellular mechanisms involved in CO2/H+ sensing by RTN neurons, and how those are established developmentally and adapted to pathological conditions, remain matters of continuing scrutiny. In two Aims, we address a
receptor-mediated mechanism of pH sensitivity, and explore gene expression patterns that support developmental and adaptive RTN function. In Aim 1, we use new mouse genetic models to identify mechanisms of pH sensitivity in RTN neurons that are mediated by proton-activated GPR4 modulation of a background K+ channel,
exploring the hypothesis that GPR4 is expressed in RTN neurons, where its intrinsic pH sensitivity leads to
inhibition of KNa1.1 (encoded by Kcnt1) to contribute to CO2 stimulation of breathing and arousal. We propose
to: [1.1] Test whether direct detection of protons by GPR4 accounts for its effects on RTN neuronal sensitivity
and CO2-stimulated breathing; [1.2] Test whether KNa1.1 (Slo2.2, Kcnt1) is a GPR4-inhibited K+ channel effector
in RTN neurons; and [1.3] Define sites of GPR4 protein expression. In Aim 2, we combine single cell RNA-Seq
with gene manipulation and developmental/physiological challenges to test the hypothesis that Phox2b expression dictates a distinct molecular signature that supports critical physiological functions of RTN neurons, and
that those gene expression patterns are malleable to developmental and physiological challenges in support of
breathing. We propose to: [2.1] Determine consequences of Phox2b depletion on the RTN neuron transcriptome;
[2.2] Determine effect of birth on RTN neuron transcriptome; and [2.3] Characterize developmental and adaptive gene regulation in RTN neurons. To accomplish these aims, we employ a variety of techniques at multiple
levels of analysis. Specifically, we combine genetic and viral approaches for RTN neuron-specific manipulation
of gene expression; perform electrophysiological and functional/behavioral studies at the cellular and organismal levels; and utilize molecular neuroanatomy and single neuron genetic analyses for phenotypic characterization and quantification of normal and adaptive gene expression profiles.
Collectively, the proposed studies will provide novel information regarding molecular and cellular mechanisms
that regulate the pH-dependent activity of RTN neurons, at critical periods during development and in response
to physiological challenge, with relevance for identifying new therapeutic targets for disorders of breathing.
Terms: <Ablation><Acids><Acute><Address><Air><Antigenic Determinants><Apnea><Arousal><Basal Transcription Factor><Basal transcription factor genes><Binding Determinants><Birth><Blood gas><Body Tissues><Brain Stem><Brainstem><Breathing><CO2><COPD><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Candidate Disease Gene><Candidate Gene><Carbon Dioxide><Carbonic Anhydride><Cas nuclease technology><Cell Body><Cell Nucleus><Cells><Central Alveolar Hypoventilation><Central Apnea><Central Sleep Apnea><Central Sleep Apnea Syndrome><Central Sleep-Disordered Breathing><Central Sleep-Disordered Breathings><Chronic><Chronic Disease><Chronic Illness><Chronic Obstruction Pulmonary Disease><Chronic Obstructive Lung Disease><Chronic Obstructive Pulmonary Disease><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Control Locus><Cot Death><Crib Death><Data><Detection><Development><Dysfunction><Electrophysiology><Electrophysiology (science)><Epitopes><Expression Signature><Functional disorder><G Protein Coupled Receptor 4><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><GPR4><GPR4 gene><Gene Action Regulation><Gene Expression><Gene Expression Profile><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic><Genetic Models><Genetic analyses><H+ element><Hydrogen Ions><Hypoventilation><In Vitro><K channel><KI mice><Knock-in Mouse><Legal patent><Life><Measures><Mediating><Medulla Oblongata><Mice><Mice Mammals><Molecular><Molecular Fingerprinting><Molecular Profiling><Murine><Mus><Mutate><Myelencephalon><Nerve Cells><Nerve Unit><Neural Cell><Neuranatomies><Neuranatomy><Neuroanatomies><Neuroanatomy><Neurocyte><Neuronal Dysfunction><Neurons><Neuropeptides><Neurophysiology / Electrophysiology><Nucleus><Ondine Syndrome><PACAP><Parturition><Patents><Pathologic><Pattern><Phenotype><Physiologic><Physiological><Physiopathology><Pons><Pons Cerebelli><Pons Varolii><Pontine><Pontine structure><Population><Potassium Channel><Potassium Ion Channels><Process><Protons><Proxy><Receptor Protein><Regulation><Respiration><Respiration Disorders><Respiratory Aspiration><Respiratory Disorder><Respiratory Inspiration><Role><SIDS><Sensory><Site><Sudden Infant Death><Sudden Unexpected Infant Death><Sudden infant death syndrome><Syndrome><System><Techniques><Testing><Time><Tissues><Transcription Factor Proto-Oncogene><Transcription factor genes><Viral><Work><base><bases><behavior study><behavioral study><breathing disorder><chronic disorder><chronic obstructive pulmonary disorder><congenital central hypoventilation syndrome><critical period><developmental><electrophysiological><excitatory neuron><experiment><experimental research><experimental study><experiments><gene expression pattern><gene expression signature><gene manipulation><genetic analysis><genetic manipulation><genetically manipulate><genetically perturb><global gene expression><global transcription profile><in vivo><inspiration><knockin mice><molecular profile><molecular signature><mouse genetics><mutant><neonatal period><neural cell body><neural dysfunction><neuronal><neuronal cell body><neuronal excitability><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><pathophysiology><perinatal period><perinatal phase><pituitary adenylate cyclase activating peptide><pituitary adenylate cyclase activating polypeptide><postnatal><premature><prematurity><protein expression><receptor><respiratory><respiratory dysfunction><respiratory mechanism><response><restoration><scRNA-seq><sensor><sensory mechanism><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><social role><soma><transcription factor><transcriptional profile><transcriptional signature><transcriptome><transcriptomics>