Stable Isotope Labelling Kinetics (SILK) tracing of neurofilament light in human cerebrospinal fluid and brain tissue

Abstract

Background

Neurofilament light protein (NfL) is a promising biomarker of neuronal injury and neurodegeneration. NfL levels in cerebrospinal fluid (CSF) and blood provide information about disease progression and are increasingly relied on as outcome measure in clinical trials. Understanding NfL kinetics in vivo is critical for interpreting NfL in response to new events where a steady state cannot be assumed, such as acute injury, disease onset or progression, or response to disease-modifying therapies.

Method

We infused human participants with diagnosed primary tauopathies (progressive supranuclear palsy, n = 5; corticobasal syndrome, n = 3; behavioural variant frontotemporal dementia, n = 2) with a stable isotope tracer (¹³C₆-leucine) and collected CSF by lumbar puncture at 4, 14, 20, 60 and 120 days post-labelling. In addition, post-mortem brain tissue from three participants who were infused with the tracer 18, 44 and 50 months earlier were homogenised and biochemically fractionated to separate the soluble and insoluble fraction. NfL was enriched in all samples via immunoprecipitation. The ratio of labelled to unlabelled NfL was measured monitoring proteotypic peptides using established targeted mass spectrometry workflows to quantitate the tracer-to-tracee ratio (TTR).

Result

Analysis of CSF detected low labelling of NfL (0.04 – 0.36% TTR) by 120 days that was comparable to the TTR levels detected in the soluble brain fraction. There was NfL present in the insoluble fraction (2.87% of the total NfL at 18-, 8.87% at 44- and 14.03% at 50-months post-mortem, averaged across several NfL peptides), with different relative abundances of NfL domains between these fractions. Interestingly, the ¹³C₆-labelled NfL signal detected in the insoluble fraction was more abundant, despite a higher recovery of total NfL in the soluble fraction.

Conclusion

NfL turnover in vivo is remarkably slow as it is scarcely captured by 120 days post-labelling. The labelling results from brain tissue suggest that in these cases of primary tauopathies newly-synthesized NfL might be delocalized to a pool of protein of low solubility that does not contribute to NfL levels in CSF. Current experiments are addressing whether newly-synthesized NfL might be sequestered into pathological inclusions.