Stable Isotope Labeling Kinetics (SILK) tracing of neurofilament light in stem cell‐derived neurons

Abstract

BackgroundNeurofilament light polypeptide (NfL) is a basic component of intermediate filaments in neurons that is released from damaged and/or degenerated neuronal axons. As such, NfL levels in CSF and serum are elevated in several neurodegenerative diseases, and have been proposed as a promising biomarker of axonal damage. However, whether NfL’s extracellular release results from passive events or active mechanisms remains largely unknown. Kinetic labeling at the step of protein translation can inform about active translation vs. passive release processes and provide protein metabolism half‐lives and rates of production and clearance.MethodOur aim was to develop a method to study NfL turnover rates using stable isotope labeling kinetics (SILK) in human induced pluripotent stem cell (iPSC)‐derived neurons. We added 13C6‐leucine, the tracer, to the neuron cultures (“pulse”) for four weeks, followed by medium exchange with 12C6‐leucine, the tracee, for an additional four weeks (“chase”). Samples were collected during both the pulse and chase phases of the experiment. 13C6‐leucine incorporation and the levels of labeled NfL in cell lysates and conditioned media from two independent neuronal inductions were analyzed by ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) and the tracer‐to‐tracee ratio (TTR) and mole percent excess (MPE) calculated.ResultWe detected ten leucine‐containing peptides unique to NfL in neuronal cell lysates, which are present within the rod‐domain of the protein. These peptides showed different rates of 13C6‐leucine incorporation (reaching up to 69% MPE).ConclusionPreliminary data on the monitoring of these peptides suggest that NfL is turned over at a slow rate in iPSC‐. Tracking 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.

Understanding and Modeling the Pathophysiology of Hydrocephalus: In Search of Better Treatment Options

“…Interestingly, the organoids can produce CSF-like fluid. When the CSF-like fluid from the organoid was compared with CSF proteins from human patients, the same proteins were present [112]. Another group made a CP organoid that had a similar morphology and expressed the genes LIM homeobox transcription factor 1 alpha (LMX1A), orthodenticle homeobox 2 (OTX2), and aquaporin 1 (AQP1) that are also expressed in the CP [102].…”