SMN Is Physiologically Downregulated at Wild-Type Motor Nerve Terminals but Aggregates Together with Neurofilaments in SMA Mouse Models

Abstract

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.

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

“…CMT-causative NEFL mutations trigger NF-L accumulation and aggregation in neurons 17 – 21 , and WT NF protein aggregation is a feature of many other neurodegenerative disorders 16 , 22 – 25 . Given our results demonstrating that O-GlcNAc influences NF-L assembly state and the many well-established connections among O-GlcNAcylation, protein aggregation, and neurodegeneration in general 43 , we tested whether NF dysfunction impacted NF-L O-GlcNAcylation.…”

“…CMT-causative mutations in NEFL or other genes result in NF aggregation and aberrant motility of neuronal mitochondria 15 – 21 , underlining the physiological importance of NF-L function. Aggregation of wild-type (WT) NF-L and other NF proteins is also a pathological hallmark of a variety of neurological conditions, including Alzheimer’s disease (AD) 22 , Parkinson’s disease (PD) 23 , amyotrophic lateral sclerosis (ALS) 24 , giant axonal neuropathy (GAN) 16 , and spinal muscular atrophy 25 . Recently, NF-L levels in the cerebrospinal fluid (CSF) and blood have emerged as powerful biomarkers of neuronal injury and nervous system disorders 2 , 26 , showing promise for early diagnosis in a variety of clinical settings 27 – 29 .…”

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

“…In particular, the NF network is required for neuronal shape, mechanical integrity, organelle trafficking, and synapse architecture [2][3][4] . In addition to their physiological importance, the dysregulation of NF proteins contributes to myriad neurological diseases 21,22,25,[31][32][33][34] . Therefore, understanding the dynamic regulation of the NF cytoskeleton is a crucial -but incompletely realized -goal.…”

“…CMT-causative NEFL mutations trigger NF-L accumulation and aggregation in neurons [26][27][28][29][30] , and WT NF protein aggregation is a feature of many neurodegenerative disorders 25,[31][32][33][34] . Given our results demonstrating that O-GlcNAc influences NF-L assembly state and the many well-established connections among O-GlcNAcylation, protein aggregation, and neurodegeneration in general 62 , we tested whether NF dysfunction impacted NF-L O-GlcNAcylation.…”

“…CMT-causative mutations in NEFL or other genes result in NF aggregation and aberrant motility of neuronal mitochondria [24][25][26][27][28][29][30] , underlining the physiological importance of NF-L function. Aggregation of wild type (WT) NF-L and other NF proteins is also a pathological hallmark of a variety of neurological conditions, including Alzheimer's disease (AD) 31 , Parkinson's disease (PD) 32 , amyotrophic lateral sclerosis (ALS) 33 , giant axonal neuropathy 25 , and spinal muscular atrophy 34 . Recently, NF-L levels in the cerebrospinal fluid (CSF) and blood have emerged as powerful biomarkers of neuronal injury and nervous system disorders 3,4, 35 , showing promise for early diagnosis in a variety of clinical settings [36][37][38][39][40] .…”