This proposal aims at elucidating the physiological, molecular and anatomical properties of cerebrospinal fluid-contacting neurons (CSF-cNs) in the mammalian central nervous system. CSF-cNs are a unique neuronal population, conserved from lower vertebrates to primates, lining the central canal (CC) at all medullospinal levels and are characterized by selective expression of the Polycystin Kidney Disease 2-Like 1 channel (PKD2L1). These neurons are highly polarized cells with a dendrite projecting to the CC and forming a large protrusion in contact with the CSF, while their axons appear to extend in the parenchyma along the longitudinal axis of the ventral spinal cord. CSF-cNs are strategically positioned to monitor CSF composition and sense mechanical deformation of the spine, thus possible functions in providing sensory information about the physiological and/or postural state of the organism have been hypothesized. In recent studies, we conducted pioneering characterization of the physiology and anatomy of mouse medullar CSF-cNs, demonstrating expression of functional ionotropic receptors and spontaneous PKD2L1 channel activity capable of regulating CSF-cNs excitability. We also described that CSF-cNs positioning around the CC varies at different segmental levels, suggesting potential differences in physiological properties, circuitry and function along the rostro-caudal axis of CNS. However, to date, the role of CSF-cNs in the central nervous system remains unknown. By combining electrophysiological, calcium imaging, molecular and viral tracing techniques, we propose to determine for the first time in mammals the molecular profile, cellular and connectivity properties of CSF-cNs at different medullospinal levels according to the following specific aims: AIM 1 - Physiology and synaptic integration of CSF-cNs along the central canal A. CSF-cNs electrophysiological properties and calcium homeostasis B. Region specific transcriptomic on isolated medullospinal CSF-cNs C. Identification of CSF-cNs input/output relationships and modulation of their activity AIM 2 - Characterization of CSF-cNs input-output connectivity A. Identification of CSF-cNs projection patterns and pre-synaptic terminals B. Mapping CSF-cNs circuits C. Identification of CSF-cNs pre- and post-synaptic partners The proposed studies will advance our current understanding of CSF-cNs biology to fully characterize their molecular and cellular properties. Moreover, we will determine both anatomically and functionally the specific neuronal networks CSF-cNs are inserted in. Altogether, these data will be a necessary step to define the function of CSF-cNs in the mammalian central nervous system.

Complement-mediated kidney diseases are ultra-rare conditions associated with genetic variants or circulating auto-antibodies, allowing uncontrolled complement activation, and resulting in complement deposition in the kidneys. This leads to renal inflammation and ultimately end-stage renal failure that can recur after renal transplantation. Here we will investigate two rare kidney diseases, atypical hemolytic uremic syndrome and C3 glomerulopathy. In both there is an unmet need for accurate testing of genetic and acquired complement abnormalities. Interlaboratory complement assays vary considerably, and their standardization is a great necessity. Solving of these challenges is needed for the understanding of disease pathophysiology and for correct diagnosis and treatment. We aim to determine appropriate biomarkers in the circulation and tissue for diagnosis using patient blood samples, DNA, blood cell-derived endothelial cells, extracellular vesicles, and kidney biopsies. With the advantage of a large patient cohort, we will standardize immunological detection assays. Furthermore, we will use advanced state-of-the-art methodology for the development of diagnostic assays for complement activation based on proteomics, flow cytometry, cell-free glycocalyx-based assays, transcriptomics and histological immunostaining. The phenotype of genetic variants found in patients will be assessed using these novel assays both on cells and intracellularly. We also aim to explore novel pathways of complement activation by renin, the kallikrein-kinin system and heme using both in vitro and in vivo models. The scientific consortium, from 6 European countries, including an early career scientist, combines expertise in adult and pediatric nephrology, immunology, genetics, basic and translational research with extensive experience in the research, diagnosis and cure of these rare kidney diseases. The consortium is associated with a specialized SME in the field of complement and several European patient organizations. The findings generated here we will provide better diagnostics, novel disease biomarkers, stratified genetic variants and offer a better understanding of disease pathophysiology. This will allow better improvement patient diagnosis and follow-up and define which patients will benefit from novel therapies.