Schwann cell pathology in spinal muscular atrophy (SMA)

The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. Historically, SMA has been characterised as a disease primarily affecting lower motor neurons. However, recent breakthroughs have revealed defects in other non-neuronal cells and tissues. In vivo analysis of peripheral nerve showed defects in Schwann cells, manifesting as abnormal myelination and delayed maturation of axo-glia interactions. The experiments in this thesis were designed to build on these observations and examine whether Schwann cell defects are intrinsic and occur as a primary result of low levels of SMN in that cell type, or rather represent a secondary consequence of pathology in neighbouring motor neurons. I initially developed a protocol to allow isolation of high-yields of purified, myelination-competent Schwann cells from ‘Taiwanese’ SMA mice. SMA-derived Schwann cells had significantly reduced SMN levels and failed to respond normally to differentiation cues. Increasing SMN levels restored myelin protein expression in Schwann cells from SMA mice. Perturbations in expression of key myelin proteins were likely due to failure of protein translation and/or stability rather than transcriptional defects. Co-cultures of healthy neurons with SMA Schwann cells revealed a significant reduction in myelination compared to cultures where wild-type Schwann cells were used. The presence of SMA Schwann cells also disrupted neurite stability. Perturbations in the expression of key extracellular matrix proteins, such as laminin α2, in SMA-derived Schwann cells suggests that Schwann cells were influencing neurite stability by modulating the composition of the extracellular matrix. Previous studies have demonstrated that low levels of SMN lead to disruption of ubiquitin homeostasis and decreased expression of ubiquitin-like modifier activating enzyme (UBA1) in the neuromuscular system, driving neuromuscular pathology via a beta-catenin dependent pathway. Label-free proteomics analysis of SMA and control Schwann cells identified 195 proteins with modified expression profiles. Bioinformatic analysis of these proteins using Ingenuity Pathway Analysis (IPA) software confirmed that major disruption of protein ubiquitination pathways was also present in Schwann cells from SMA mice. Immunolabeling and proteomics data both revealed that UBA1 levels were significantly reduced in SMA-derived Schwann cells. However, loss of UBA1 in Schwann cells did not lead to downstream modifications in beta-catenin pathways. Pharmacological inhibition of UBA1 in healthy Schwann cells was sufficient to induce defects in myelin protein expression, suggesting that UBA1 defects contribute directly to Schwann cell disruption in SMA. I conclude that low levels of SMN induce intrinsic defects in Schwann cells, mediated at least in part through disruption to ubiquitination pathways

Label-Free Quantitative Proteomic Profiling Identifies Disruption of Ubiquitin Homeostasis As a Key Driver of Schwann Cell Defects in Spinal Muscular Atrophy

Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues

Importance of axon-glial interactions for the normal postnatal development of the mouse peripheral nervous system

The mouse nervous system undergoes a vast remodelling of synaptic connections postnatally, resulting in a reduced number of innervating axons to target cells within the first few weeks of life. This extensive loss of connections is known as synapse elimination and it plays a critical role in sculpting and refining neural connectivity throughout the nervous system, resulting in a finely tuned and well-synchronised network of innervation. This process has been well characterised at the mouse neuromuscular junction (NMJ), where synapse elimination takes place postnatally in all skeletal muscles. It has been well studied for the reasons that it is easily accessible for live imaging and post-mortem experimental analysis. Studies utilising this synapse to uncover regulators of synapse elimination have mainly focused on the importance of glial cell lysosomal activity, nerve conduction and target-derived growth factor supply. It is clear that non-axonal cell types play key roles in the success of developmental axon retraction at the NMJ, however the role of glial cells in the regulation of this process has not been fully explored, as lysosomal activity is thought of as a consequence of axon pruning rather than a molecular driver. Previous studies have shown that signals emanating from myelinating glial cells can modulate neurofilament composition and transport within the underlying axons. We know that these changes in neurofilament composition and transport are underway during developmental synapse elimination at the NMJ, so it seems logical to predict that myelinating glial cells may play a role in the regulation of axonal pruning. Myelinating glial cells are found along the entire length of lower motor neurons and form physical interactions with the underlying axons at regions known as paranodes. At the paranode, Neurofascin155 (Nfasc155: expressed by the myelinating glial cell) interacts with a Caspr/contactin complex (expressed by the axon). This site has been proposed as a likely site for axon-glial signalling due to the close apposition of the cell membranes. The main focus of this PhD project was to study the potential role of myelinating glial cells in the success of synapse elimination at the NMJ, using a mouse model of paranodal disruption (Nfasc155-/-). Chapters 3 and 4 show the results of this work. This work has revealed a novel role for glia in the modulation of synapse elimination at the mouse neuromuscular junction, mediated by Nfasc155 in the myelinating Schwann cell. Synapse elimination was profoundly delayed in Nfasc155-/- mice and was found to be associated with a non-canonical role for Nfasc155, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Loss of Nfasc155 was sufficient to disrupt axonal proteins contributing to cytoskeletal organisation and trafficking pathways in peripheral nerve of Nfasc155-/- mice and lower levels of neurofilament light (NF-L) protein in maturing motor axon terminals. Synapse elimination was delayed in mice lacking NF-L, suggesting that Nfasc155 influences neuronal remodelling, at least in part, by modifying cytoskeletal dynamics in motor neurons. This work provides the first clear evidence for myelinating Schwann cells acting as drivers of synapse elimination, with Nfasc155 playing a critical role in glial cell-mediated postnatal sculpting of neuronal connectivity in the peripheral nervous system. A small section of the results within this thesis are devoted to the study of axon-glial interactions in a mouse model of childhood motor neuron disease, otherwise known as spinal muscular atrophy (SMA). In SMA, there are reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein. The NMJ is a particularly vulnerable target in SMA, manifesting as a breakdown of neuromuscular connectivity and progressive motor impairment. Recent studies have begun to shed light on the role of non-neuronal cell types in the onset and progression of the disease, presenting SMA as a multi-system disease rather than a purely neuronal disorder. Recent evidence has highlighted that myelinating glial cells are significantly affected in a mouse model of SMA, manifesting as an impaired ability to produce key myelin proteins, resulting in deficient myelination. The final results chapter of this thesis (Chapter 5) is focussed on further exploring the effects that loss of SMN has in Schwann cells including their interactions with underlying axons. This work reveals a disruption to axon-glial interaction, shown by a delay in the development of paranodes, supporting the idea that non-neuronal cell types are also affected in SMA. The results within this thesis reveal a novel role for a glial cell protein, Nfasc155, in the modulation of synapse elimination at the NMJ. Mechanistic insight in to Nfasc155’s role in this process is also uncovered and likely involves axonal cytoskeletal transport systems and the filamentous protein NF-L, which have not previously been implicated in the process of synapse elimination. This work highlights an important role for axon-glial interactions during normal postnatal development of the mouse NMJ. This work also highlights a role for axon-glial interactions in disease states of the NMJ. Using a mouse model of SMA, axon-glial interaction was assessed with the finding of a delay in paranodal maturation due to loss of SMN