Uman Type NF-L Antibodies Are Effective Reagents for the Imaging of Neurodegeneration

Abstract

Recent work shows that certain immunological assays for the neurofilament light chain NF-L detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to NF-L, UD1 and UD2, also known as 2.1 and 47.3 respectively. These are the essential components of the Uman Diagnostics NF-Light ELISA kit, the Quanterix Simoa bead based NF-L assay and others. We show here that the antibodies bind to neighboring epitopes in a short, conserved and unusual peptide in the NF-L "rod" Coil 2 region. We also describe a surprising and useful feature of Uman and similar reagents. While other well characterized NF-L antibodies show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of presumably spontaneously degenerating or degenerated neurons and their processes. However following experimental mid-cervical injuries to rat spinal cord both Uman antibodies recognize numerous profiles in tissue sections. The Uman positive material was associated with fiber tracts expected to be damaged by the injury administered and the profiles had the swollen, beaded, discontinuous and sinusoidal morphology expected for degenerating and degenerated processes. We also found that several antibodies to the C terminal "tail" region of NF-L stain undamaged axonal profiles but fail to recognize the Uman positive material. The unmasking of the Uman epitopes and the loss of the NF-L tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunological changes we have discovered are due to neurodegeneration induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibody reagents directed against the region of NF-L including the Uman epitopes which have staining properties identical to the Uman reagents. Using these we show that the NF-L region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the NF-L biomarker, describes novel and useful properties of Uman type and NF-L tail binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

Remyelination protects neurons from DLK-mediated neurodegeneration

“…These levels fell statistically in Myrf ΔiPlp1 mice by 20 weeks post tamoxifen, indicating that remyelination is temporally associated with a decrease in active axonal damage (Figure 4D). The staining of axons with an antibody against an epitope of neurofilament that is only exposed following proteolysis 58 further supports presence of axonal damage in Myrf ΔiSox10 mice (Figure 4E).…”

Adolescent oligodendrogenesis and myelination restrict experience-dependent neuronal plasticity in adult visual cortex

“…Myelination has long been hypothesized to be important for regulating cortical neuronal maturation and plasticity ( 2, 5, 13, 14 ), but the functional consequence of preventing developmental oligodendrogenesis and myelination has not been directly tested. We first verified that the loss of adolescent oligodendrogenesis does not induce neurodegeneration in visual cortex by immunostaining with a neurofilament light chain (NF-L) antibody that binds an epitope of NF-L accessible only in degenerating axons ( 15 ). As a positive control, we detected axonal degeneration in the spinal cord of mice that underwent experimental autoimmune encephalitis, particularly in regions with myelin loss (Figure S6A).…”

Challenges and Opportunities for the Promising Biomarker Blood Neurofilament Light Chain

“…In addition, those specific epitopes are only accessible following neurodegeneration-related protease activity. 1 Some of these issues are relatively straightforward to resolve. Although comparative studies across platforms using unified standardized biobanking procedures have yielded good consistency, additional work will be needed as new assay systems emerge.…”

“…In this regard, the general scientific community is still unsure whether measured values reflect intact protein or peptide fragments that can be influenced by sample matrix or prolonged storage. A recent preprint article raised the possibility that the antibody pair used in some of the commercially available assays bind specifically to NfL fragments from the rod construct. In addition, those specific epitopes are only accessible following neurodegeneration-related protease activity …”

“…A recent preprint article raised the possibility that the antibody pair used in some of the commercially available assays bind specifically to NfL fragments from the rod construct. In addition, those specific epitopes are only accessible following neurodegeneration-related protease activity …”