Translational profiling of bladder sensory nerves and their cell type identities using dissociation free single nucleus sequencing

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Abstract Peripheral axons need sustained maintenance and repair to maintain connectivity with their targets. Local axonal translation plays a role in regulation of protein and energy homeostasis across lifespan. The repertoire of active mRNA translation during development and adulthood, and the ability to repair, regenerate and translate specific subsets of mRNAs in relation to environmental cues is stored in distal axons. In many instances these activities take place rapidly for immediate response at target sites before new transcripts localize from the cell bodies. In disease states, altered axonal translation may affect elongation, withdrawal, pruning, branching or survival. In the bladder innervation by afferent (sensory) and efferent (parasympathetic and sympathetic) nerves is critical for its function. These nerves establish intricate connections with different regions of the bladder and cell types including the urothelium, resident immune cells, smooth muscle cells and blood vessels and likely employ local translation to maintain homeostasis and respond to injury or physiologic changes. The identity of genes that are actively translated in bladder nerve terminal and subsequently their role in bladder has not been studied. Defining actively translated mRNA in healthy and disease states in bladder nerves and the identity of the soma source will provide insights into the mechanisms of neuroanatomical and physiological interactions between the peripheral nervous and urinary systems, their roles in a number of bladder diseases with neuronal basis and aid in rationally designing targeted therapies aimed at preserving neuro-uro connections. This has potential to impact understanding of declining bladder function in ageing, debilitating bladder symptoms and dysfunction (such as intractable pain, incontinence, frequency and urgency to urinate) that affect millions of people in the US, neurodegenerative diseases and neuropathies (diabetes, HIV) affecting bladder function. We will establish methods and proof of concept studies to delineate the translatome in bladder sensory, autonomic axons in healthy male and female mice and in female mice with UTI (Aim 1) and identify their cognate cell bodies by developing artifact –free single nucleus RNA expression maps of DRG sensory neurons projecting to the bladder (Aim 2). The methods developed will overcome challenges unique to composition, function and anatomy of bladder, its nerves and corresponding soma that are different from the CNS or somatosensory system and serve as benchmark for application to other lower urinary tract organs. The methods developed, genetic tools used and novel data generated will be publicly shared for rapid adoption and accelerating studies to better understand regulation of nerve terminals with their target sites by diverse set of users including basic and translational researchers, computational biologists and clinicians interested in urinary tract disorders and normal physiology.