CSF protein clearance impairment revealed using stable isotope kinetics in normal pressure hydrocephalus

Normal pressure hydrocephalus is a common cause of gait and cognitive impairment in older adults, marked by excessive CSF accumulation. Genetic studies suggest impaired fluid clearance, and clinical symptoms can improve after CSF diversion. However, no fluid biomarkers exist to explore CSF accumulation mechanisms, assist diagnosis, or predict response to treatment. Stable isotope labelling kinetics is a clinical research tool that uses non-radioactive isotopes to label newly translated proteins, enabling measurement of their appearance (synthesis) and disappearance (clearance) in compartments like CSF. This study aimed to develop a novel method to capture protein turnover in CSF and assess whether clearance disruption is evident in normal pressure hydrocephalus with extended follow-up. Proteins of interest were identified via mass spectrometry in human CSF and choroid plexus organoid-derived CSF-like fluid. Protein origin and synthesis rates were evaluated by labelling organoids with ¹³C₆-leucine. Label incorporation was measured using targeted mass spectrometry to determine the ratio of labelled to unlabelled peptide. A proof-of-concept case-control study was then conducted in specialist neuroscience centres. Participants received intravenous ¹³C₆-leucine and underwent serial CSF withdrawal via lumbar drain, with matched blood sampling for up to 72 h. Patients undergoing CSF drainage and controls were recruited sequentially. Targeted mass spectrometry was used to determine protein production and clearance rates. To determine the clinical relevance of these protein turnover rates to CSF flow, they were correlated with direct measurements of CSF production captured using a LiquoGuard machine linked to the lumbar CSF drain. We captured choroid plexus protein kinetics in human organoids and the CSF of participants undergoing CSF drainage (n = 10) or controls (ventricular CSF n = 4; lumbar CSF n = 5). The case and control cohorts varied in sex (NPH = 80% male and controls = 22% male) and in age. There was no significant age difference between NPH and the lumbar control cohort (n = 5) (NPH: 75 (71-78) versus 70 (63-84) years old; P = 0.2438). We found that transthyretin is abundantly secreted by choroid plexus organoids, and observed correlations with CSF transthyretin synthesis rates and volume of CSF production in vivo (P = 0.738; P < 0.05). Clearance rates of transthyretin are ∼10 fold slower in normal pressure hydrocephalus compared with controls, suggesting impaired CSF protein clearance. This method is a novel clinical tool for interrogating CSF protein dynamics and may have utility in tracking CSF flow clinically.