Synaptic Neurofilaments and GluN1-Neurofilament Light Chain Interaction in Experimental Models of α-Synucleinopathies

Abstract

Introduction: despite neurofilaments are mainly expressed in large caliber myelinated axons, recent evidence supports the existence of a specific synaptic pool, where neurofilament light chain (NfL) has been proposed to stabilize NMDAR at post-synaptic membrane through a direct interaction with the GluN1 subunit. Here, we assessed the expression and synaptic abundance of neurofilaments and their interaction with NMDAR in experimental α-synucleinopathy models. Methods: we used confocal imaging and biochemical approaches to confirm NMDAR-NfL interaction at synapses. Western blotting in purified fractions and Co-IP were then performed to assess synaptic neurofilaments expression and GluN1-NfL interaction in (i) α-synuclein pre-formed fibrils- (α-syn PFF) treated hippocampal neuronal cultures and (ii) mice intrastriatally injected with α-syn-PFF. Results: we identified the existence of a direct protein-protein interaction between NMDAR and NfL endogenously expressed in neurons. Our findings showed increased striatal GluN1-NfL interaction levels at early phases of α-syn PFF-treated mice compared to controls (NfL/GluN1 optical density: α-syn PFF 0.71 ± 0.04; controls 0.48 ± 0.03; t(9) = 4.67; p = 0.001). In agreement with this observation, we found that NfL levels are increased in striatal post-synaptic fractions of α-syn PFF-treated mice (normalized optical density: α-syn PFF 1.86 ± 0.14; controls 1.34 ± 0.13 t(18) = 2.70; p = 0.015). Conclusions: our results demonstrate alterations of striatal synaptic neurofilament pool in α-synucleinopathy models and open the way to further investigations evaluating a potential role of neurofilaments dysregulation in explaining glutamatergic synaptic dysfunction observed in α-synucleinopathies such as Parkinson’s disease.

Synaptic status differentially regulates neurofilaments in dendritic spines

“…Together with the demonstration of isoform-specific interaction with receptors at synapses (e.g. NFL and NMDA receptors, NFM and D1 dopamine receptors, [10,12,19]), this data points at isoform-specific functions of NFs within the context of the postsynapses.…”

“…For a long time NFs have been considered as mere space-fillers in the axon, albeit present also in dendrites [6][7][8]. Only in the last years a link between NF function in synapses and psychiatric disorders started to emerge [9][10][11][12]. Indeed, NF were identified at pre-and postsynaptic sites of excitatory and inhibitory synapses where they have a different phosphorylation status compared to the overall NF population [13][14][15].…”

“…Inhibition of NFH was shown to depress long-term potentiation (LTP) in the hippocampus without altering the spines morphology, which, on the other hand, is affected in NFL knockout animals [13,16]. Furthermore, NFM functionally interacts with dopamine D1-receptors, while both NFL and α-internexin regulate the localization of N-methyl-D-aspartate (NMDA) receptors subunits, providing direct evidence for the involvement of NFs in the regulation of synaptic functions [12,13,[16][17][18][19]. However, whether and how the NF composition of a spine correlates with the synaptic status, and in particular the presynaptic activity and the postsynaptic strength, is unclear.…”

O-GlcNAcylation regulates neurofilament-light assembly and function and is perturbed by Charcot-Marie-Tooth disease mutations

“…Of the triplet proteins, NF-light (NF-L) is required for the structural integrity of axons [13][14][15][16] , has been detected at post-synaptic sites 17 , and directly interacts with the N-methyl-D-aspartate receptor to influence high-order brain functions [18][19][20] . Ablating the NEFL gene, which encodes NF-L, impairs maturation of regenerating myelinated axons 13 , dendritic arborization 14 , and peripheral nerve regeneration in mice 15 .…”

Neurofilament Levels in Dendritic Spines Associate with Synaptic Status

“…Traditionally, NFs have been considered as structural components in the axon, albeit also present in dendrites and synaptosomes [ 6 , 7 , 8 , 9 ]. Although the role of NFs in signal transduction was proposed decades ago [ 10 , 11 , 12 ], a link between NF function in synapses and synaptic disorders has started to emerge only in the last years [ 13 , 14 , 15 , 16 ]. Indeed, NFs were identified at pre- and postsynaptic sites of excitatory and inhibitory synapses in the form of short 9–10 nm long filaments corresponding to oligomeric structures [ 17 , 18 ].…”