Motor unit recovery following Smn restoration in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a childhood motor neuron disease caused by anomalies in the SMN1 gene. Although therapeutics have been approved for the treatment of SMA, there is a therapeutic time window, after which efficacy is reduced. Hallmarks of motor unit pathology in SMA include loss of motor-neurons and neuromuscular junction (NMJs). Following an increase in Smn levels, it is unclear how much damage can be repaired and the degree to which normal connections are re-established. Here, we perform a detailed analysis of motor unit pathology before and after restoration of Smn levels. Using a Smn-inducible mouse model of SMA, we show that genetic restoration of Smn results in a dramatic reduction in NMJ pathology, with restoration of innervation patterns, preservation of axon and endplate number and normalized expression of P53-associated transcripts. Notably, presynaptic swelling and elevated Pmaip levels remained. We analysed the effect of either early or delayed treated of an antisense oligonucleotide (ASO) targeting SMN2 on a range of differentially vulnerable muscles. Following ASO administration, the majority of endplates appeared fully occupied. However, there was an underlying loss of axons and endplates, which was more prevalent following a delay in treatment. There was an increase in average motor unit size following both early and delayed treatment. Together this work demonstrates the remarkably regenerative capacity of the motor neuron following Smn restoration, but highlights that recovery is incomplete. This work suggests that there is an opportunity to enhance neuromuscular junction recovery following administration of Smn-enhancing therapeutics.

LCM-seq reveals unique transcriptional adaptation mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy.

Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.

Neurturin is a PGC-1α1-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation.

OBJECTIVE: We examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. METHODS: A microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. RESULTS: Skeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. CONCLUSIONS: Our findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons.

Motor neuron vulnerability and resistance in amyotrophic lateral sclerosis.

In the fatal disease-amyotrophic lateral sclerosis (ALS)-upper (corticospinal) motor neurons (MNs) and lower somatic MNs, which innervate voluntary muscles, degenerate. Importantly, certain lower MN subgroups are relatively resistant to degeneration, even though pathogenic proteins are typically ubiquitously expressed. Ocular MNs (OMNs), including the oculomotor, trochlear and abducens nuclei (CNIII, IV and VI), which regulate eye movement, persist throughout the disease. Consequently, eye-tracking devices are used to enable paralysed ALS patients (who can no longer speak) to communicate. Additionally, there is a gradient of vulnerability among spinal MNs. Those innervating fast-twitch muscle are most severely affected and degenerate first. MNs innervating slow-twitch muscle can compensate temporarily for the loss of their neighbours by re-innervating denervated muscle until later in disease these too degenerate. The resistant OMNs and the associated extraocular muscles (EOMs) are anatomically and functionally very different from other motor units. The EOMs have a unique set of myosin heavy chains, placing them outside the classical characterization spectrum of all skeletal muscle. Moreover, EOMs have multiple neuromuscular innervation sites per single myofibre. Spinal fast and slow motor units show differences in their dendritic arborisations and the number of myofibres they innervate. These motor units also differ in their functionality and excitability. Identifying the molecular basis of cell-intrinsic pathways that are differentially activated between resistant and vulnerable MNs could reveal mechanisms of selective neuronal resistance, degeneration and regeneration and lead to therapies preventing progressive MN loss in ALS. Illustrating this, overexpression of OMN-enriched genes in spinal MNs, as well as suppression of fast spinal MN-enriched genes can increase the lifespan of ALS mice. Here, we discuss the pattern of lower MN degeneration in ALS and review the current literature on OMN resistance in ALS and differential spinal MN vulnerability. We also reflect upon the non-cell autonomous components that are involved in lower MN degeneration in ALS.

Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3ß phosphorylation and ß-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

Cross-disease comparison of amyotrophic lateral sclerosis and spinal muscular atrophy reveals conservation of selective vulnerability but differential neuromuscular junction pathology.

Neuromuscular junctions are primary pathological targets in the lethal motor neuron diseases spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Synaptic pathology and denervation of target muscle fibers has been reported prior to the appearance of clinical symptoms in mouse models of both diseases, suggesting that neuromuscular junctions are highly vulnerable from the very early stages, and are a key target for therapeutic intervention. Here we examined neuromuscular pathology longitudinally in three clinically relevant muscle groups in mouse models of ALS and SMA in order to assess their relative vulnerabilities. We show for the first time that neuromuscular junctions of the extraocular muscles (responsible for the control of eye movement) were resistant to degeneration in endstage SMA mice, as well as in late symptomatic ALS mice. Tongue muscle neuromuscular junctions were also spared in both animal models. Conversely, neuromuscular junctions of the lumbrical muscles of the hind-paw were vulnerable in both SMA and ALS, with a loss of neuronal innervation and shrinkage of motor endplates in both diseases. Thus, the pattern of selective vulnerability was conserved across these two models of motor neuron disease. However, the first evidence of neuromuscular pathology occurred at different timepoints of disease progression, with much earlier evidence of presynaptic involvement in ALS, progressing to changes on the postsynaptic side. Conversely, in SMA changes appeared concomitantly at the neuromuscular junction, suggesting that mechanisms of neuromuscular disruption are distinct in these diseases. J. Comp. Neurol. 524:1424-1442, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Reversible molecular pathology of skeletal muscle in spinal muscular atrophy.

Low levels of full-length survival motor neuron (SMN) protein cause the motor neuron disease, spinal muscular atrophy (SMA). Although motor neurons undoubtedly contribute directly to SMA pathogenesis, the role of muscle is less clear. We demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic severe SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomic data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. Robust upregulation of voltage-dependent anion-selective channel protein 2 (Vdac2) and downregulation of parvalbumin in severe SMA mice was confirmed in a milder SMA mouse model and in human patient muscle biopsies. Molecular pathology of skeletal muscle was ameliorated in mice treated with the FDA-approved histone deacetylase inhibitor, suberoylanilide hydroxamic acid. We conclude that intrinsic pathology of skeletal muscle is an important and reversible event in SMA and also suggest that muscle proteins have the potential to act as novel biomarkers in SMA.

ApoE isoform-specific regulation of regeneration in the peripheral nervous system.

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood-nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo.

Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research.

BACKGROUND: Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that thy1-driven expression of yellow fluorescent protein (YFP) in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.

The response of neuromuscular junctions to injury is developmentally regulated.

It is well established that developmental maturity is a key factor regulating the response of lower motor neurons to injury. The influence of age on the survival of motor neuron cell somata following axotomy is well documented, but it remains unclear whether maturity also influences the degeneration of axonal and synaptic compartments at the neuromuscular junction. Such information is important for our interpretation of data suggesting that neonatal neuromuscular junctions are particularly vulnerable in neurodegenerative conditions that affect the developing postnatal nervous system, such as spinal muscular atrophy. Here, we have examined the role of development in regulating the vulnerability of mouse neuromuscular junctions to two mechanistically distinct degenerative insults: hypoxia and peripheral nerve injury. We report that neuromuscular junctions in neonatal mice are significantly more resistant to both hypoxia and nerve injury than those in adult mice, with a transition from the neonatal to adult phenotype occurring at 2-3 wk of age. We also demonstrate that the reduced vulnerability of neuromuscular junctions observed in neonatal mice is not determined by the maturity of the synapse per se, suggesting that properties associated with the neonatal environment and/or age of the neuron are responsible for modulating vulnerability. Our results are in stark contrast to previous studies showing that motor neuron cell somata are markedly more vulnerable to axotomy in neonatal mice. We conclude that neonatal neuromuscular junctions are resistant to a range of neurodegenerative insults in vivo and that this resistance is developmentally regulated.

Age-related motor neuron degeneration in DNA repair-deficient Ercc1 mice.

Degeneration of motor neurons contributes to senescence-associated loss of muscle function and underlies human neurodegenerative conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors contributing to motor neuron vulnerability and degenerative phenotypes in vivo are therefore important for our understanding of the neuromuscular system in health and disease. Here, we analyzed neurodegenerative abnormalities in the spinal cord of progeroid Ercc1(Delta/-) mice that are impaired in several DNA repair systems, i.e. nucleotide excision repair, interstrand crosslink repair, and double strand break repair. Ercc1(Delta/-) mice develop age-dependent motor abnormalities, and have a shortened life span of 6-7 months. Pathologically, Ercc1(Delta/-) mice develop widespread astrocytosis and microgliosis, and motor neuron loss and denervation of skeletal muscle fibers. Degenerating motor neurons in many occasions expressed genotoxic-responsive transcription factors p53 or ATF3, and in addition, displayed a range of Golgi apparatus abnormalities. Furthermore, Ercc1(Delta/-) motor neurons developed perikaryal and axonal intermediate filament abnormalities reminiscent of cytoskeletal pathology observed in aging spinal cord. Our findings support the notion that accumulation of DNA damage and genotoxic stress may contribute to neuronal aging and motor neuron vulnerability in human neuromuscular disorders.

Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and post-synaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here we have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/-;SMN2 and Smn-/-;SMN2;Delta7 mouse models of SMA. We show that neuromuscular junctions in the transversus abdominis (TVA), levator auris longus (LAL) and lumbrical muscles were disrupted in both mouse models. Pre-synaptic inputs were lost and abnormal accumulations of neurofilament were present, even in early/mid-symptomatic animals in the most severely affected muscle groups. Neuromuscular pathology was more extensive in the postural TVA muscle compared with the fast-twitch LAL and lumbrical muscles. Pre-synaptic pathology in Smn-/-;SMN2;Delta7 mice was reduced compared with Smn-/-;SMN2 mice at late-symptomatic time-points, although post-synaptic pathology was equally severe. We demonstrate that shrinkage of motor endplates does not correlate with loss of motor nerve terminals, signifying that one can occur in the absence of the other. We also demonstrate selective vulnerability of a subpopulation of motor neurons in the caudal muscle band of the LAL. Paralysis with botulinum toxin resulted in less terminal sprouting and ectopic synapse formation in the caudal band compared with the rostral band, suggesting that motor units conforming to a Fast Synapsing (FaSyn) phenotype are likely to be more vulnerable than those with a Delayed Synapsing (DeSyn) phenotype.