Stable Isotope Labelling Kinetics Tracing of Neurofilament Light in Stem Cell‐derived Neurons and Humans

AbstractBackgroundNeurofilament light (NfL) is a neuronal cytoskeletal protein that is released into the extracellular environment from degenerated axons. As such, NfL levels in cerebrospinal fluid (CSF) and serum have been proposed as a promising biomarker of neurodegeneration, yet the metabolism of NfL in human neurons is unknown. Kinetic protein labelling can provide protein half‐lives and rates of production and clearance in vitro and in vivo.MethodUsing a stable isotope tracer (13C6‐leucine), we labelled two lines of pluripotent stem cell‐derived neurons and human study participants with primary tauopathies (n = 9). We collected neuronal lysates and conditioned media every three days during the labelling (“pulse”) and post‐labelling (“chase”) phases. CSF samples were collected across five visits, scheduled around 4, 14, 20, 60 and 120 days post‐labelling. We measured the ratio of labelled to unlabelled NfL using targeted mass spectrometry workflows to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), together providing the protein turnover rate.ResultThe average half‐life of NfL in cell lysates was four days (± 1.06 SD, n = 3 independent biological replicates). The turnover of extracellular NfL in neuronal conditioned media was slower than that found in neuronal lysates, suggesting that NfL is a rather stable protein in the extracellular milieu in vitro. In contrast, analysis of CSF from the in vivo tauopathy cohort detected low labelling of NfL (0.04 – 0.36%) by 120 days (n = 5), indicating that in vivo turnover is exceptionally slower than in vitro and that extended CSF collection and analysis >120 days post‐labelling is crucial to capture NfL production and clearance in vivo. To this end, additional analysis of healthy controls and patients with Alzheimer’s disease (control and AD cohorts; n = 24), which include CSF collection up to 219 ‐ 444 days post‐labelling, is ongoing.ConclusionTracking of both intracellular and extracellular NfL in neurons derived from healthy control individuals, as well as cells with mutations implicated in neurodegenerative diseases, are expected to shed light into the dynamics of NfL turnover in both physiological and pathological conditions. This will be critical for interpreting NfL in response to disease‐modifying therapies.