Schwann cell pathology in spinal muscular atrophy (SMA)
Aghamaleky Sarvestany, Arwin
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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.