Prion-like transmission of α-synuclein pathology in the context of an NFL null background

Abstract

Neurofilaments are a major component of the axonal cytoskeleton in neurons and have been implicated in a number of neurodegenerative diseases due to their presence within characteristic pathological inclusions. Their contributions to these diseases are not yet fully understood, but previous studies investigated the effects of ablating the obligate subunit of neurofilaments, low molecular mass neurofilament subunit (NFL), on disease phenotypes in transgenic mouse models of Alzheimer's disease and tauopathy. Here, we tested the effects of ablating NFL in α-synuclein M83 transgenic mice expressing the human pathogenic A53T mutation, by breeding them onto an NFL null background. The induction and spread of α-synuclein inclusion pathology was triggered by the injection of preformed α-synuclein fibrils into the gastrocnemius muscle or hippocampus in M83 versus M83/NFL null mice. We observed no difference in the post-injection time to motor-impairment and paralysis endpoint or amount and distribution of α-synuclein inclusion pathology in the muscle injected M83 and M83/NFL null mice. Hippocampal injected M83/NFL null mice displayed subtle region-specific differences in the amount of α-synuclein inclusions however, pathology was observed in the same regions as the M83 mice. Overall, we observed only minor differences in the induction and transmission of α-synuclein pathology in these induced models of synucleinopathy in the presence or absence of NFL. This suggests that NFL and neurofilaments do not play a major role in influencing the induction and transmission of α-synuclein aggregation.

Keywords: NFL gene (NEFL) knockout; Neurofilaments; Parkinson’s disease; Synucleinopathy; Transgenic mice; α-Synuclein.

Figure 1. Generation of M83 transgenic mice on an NFL null background

(A) Genotyping analyses to confirm the presence of the SNCA transgene and absence of NEFL. Sizes are in base pairs. (B) Western blot analysis of total protein lysates from spinal cord of wild-type (WT), M83, NFL null and M83/NFL null mice. Antibody NR4 shows the presence/absence of NFL. Antibody SNL1 shows the overexpression of αS. GAPDH and β-actin are shown as loading controls. Molecular weights are in kDa.

Figure 2. Assessment of motor impairment and αS inclusion pathology of M83/NFL null mice versus M83 mice following intramuscular injection of αS fibrils

(A) Survival curve of M83 and M83/NFL null mice following intramuscular injection of mouse αS fibrils (n=7 and 9, respectively) or PBS (n=10; M83/NFL null mice only). Curves of mouse αS fibril injected mice are not significantly different (Log-rank test; p=0.662). (B) Distribution maps of LS4-2G12 immunopositive αS pathology in mouse αS fibril muscle injected M83 and M83/NFL null mice. (C) Representative images of immunohistochemistry staining of αS pathology in the spinal cord, brainstem and thalamus of mouse αS fibril muscle injected M83 and M83/NFL null mice. LS4-2G12 detects pSer129 αS, 9C10 detects the N-terminus of αS and p62 is a general inclusion marker. Scale bars = 100μm.

Figure 3. Hippocampal αS pathology in M83/NFL null mice versus M83 mice following intrahippocampal injection of αS fibrils

(A) Representative images of immunohistochemistry staining of hippocampi of M83 and M83/NFL null mice 4 months following intrahippocampal injection of human αS fibrils. Scale bar = 300μm. (B) Distribution maps of LS4-2G12 immunopositive αS inclusion pathology in hippocampal injected M83 and M83/NFL null mice (n=10/group).

Figure 4. Representative αS inclusion pathology in M83/NFL null mice versus M83 mice following intrahippocampal injection of αS fibrils

(A) Representative images of immunohistochemistry staining of the entorhinal cortex, thalamus, brainstem and spinal cord of M83 and M83/NFL null mice 4 months following intrahippocampal injection of human αS fibrils. Scale bars = 100μm.