Identifying mechanisms of neurofilament regulation and turnover in amyotrophic lateral sclerosis

Program Summary Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by loss of motor neurons that leads to weakness, respiratory failure, and death within 3-5-years of symptom onset. The importance of prognostic and pharmacodynamic biomarkers in therapeutic development is highlighted by the emergence of neurofilament light

(NfL) and phosphorylated neurofilament heavy (pNfH) as potential neurodegenerative biomarkers for ALS. Neurofilaments (NFs) are represented by three subunits: NfL, neurofilament medium (NfM), and NfH that complex with -internexin in the central nervous system (CNS) or peripherin in the peripheral nervous system (PNS). NFs undergo extensive post-translational

modifications (PTMs) (i.e. phosphorylation, O-glycosylation) that regulate neurofilament assembly, transport, and function and are known to form pathologic aggregates in ALS. An antisense oligonucleotide to SOD1, tofersen, was recently granted accelerated approval for hereditary SOD1-ALS based on its ability to lower NfL and pNfH by immunoassay in serum and

CSF by ~60% at 12 weeks, long before clinical improvement was observed at one year. However, immunoassay methods are vulnerable to non-specific signals and are unable to discriminate between alternative isoforms or PTMs that may occur with disease. We have developed a proteomic assay for NfL that has indicated NfL exists only as truncated fragments

in ALS CSF and have found that Coil 1 domain peptide species correlate best with ALS disease progression. We have also developed reagents and methods to extend analysis to NfM and NfH. By comparing neurofilament (NF) species in ALS and control biofluids, we anticipate that we will identify NF isoforms and PTMs unique to ALS. We will also measure NF isoforms pre-

and post- tofersen treatment in blood and CSF from SOD1-ALS participants and compare their performance to existing NfL and pNfH immunoassays. We recently demonstrated that stable isotope labelling kinetics (SILK) can be safely employed in ALS participants and showed that mutant SOD1A5V protein turnover is faster than its wild-type counterpart. In this study, we will

examine the effect of SOD1 lowering therapy on neuronal proteins, tau and NfL, and perform proteomic analysis to assess changes in protein expression pre- and post-treatment. We propose that in-depth proteomic and protein kinetic analysis of biofluids from the tofersen treated SOD1-ALS population provides an unparalleled opportunity to uncover biomarkers

related to clinical improvement in ALS.