Murray, Lyndsay

Smillie, Karen

Simmen, Martin

Woschitz, Victoria

2023-11-14T12:30:10Z

2023-11-14T12:30:10Z

2023-11-14

Spinal muscular atrophy (SMA) is a childhood form of motor neuron disease. It is characterised by the loss of lower motor neurons and muscle weakening and atrophy of associated muscles. Motor neurons (MNs) are the primary pathological target of SMA, but it has long been shown that breakdown of neuromuscular junctions (NMJs) is also an early pathological target of the disease. However, not all MNs are equally vulnerable, with some being lost very early in the course of the disease, whilst other subpopulations are consistently spared until late stages of the disease. This selective vulnerability of MNs is a well-known pathological hallmark of SMA, however the underlying molecular mechanisms of it are yet unknown. The work contained within this thesis aims to investigate the basis of selective vulnerability, which will give critical insight into the factors which drive motor neuron pathology and help identify novel neuroprotective strategies. Selective vulnerability of MNs and a wide range of muscles has been reported in SMA patients and in commonly utilised mouse models of SMA. Despite the many publications in each mouse model of SMA, there has not yet been a comparison of NMJ pathology across different mouse models of SMA. I therefore performed an extensive analysis of NMJ pathology in 20 muscles from the Smn²ᴮ/⁻ mouse model of SMA and subsequently compared pattern of selective vulnerability between other mouse models of SMA. This revealed a more profound motor neuron cell body loss in thoracic and upper lumbar spinal cord segments and higher levels of NMJ loss in muscles innervated by nerves from these spinal cord segments in the Smn²ᴮ/⁻ mouse model. Comparison of selective vulnerability patterns between the Smn²ᴮ/⁻ mouse model and other mouse models of SMA, showed that all models had a distinct pattern of selective vulnerability, with the Smn²ᴮ/⁻ showing most similarity to patients. Patterns of vulnerability could not be attributed to developmental subtype or muscle fibre type in any of the mouse models. To gain insight into the molecular mechanisms regulating selective vulnerability, I performed cross-model transcriptional analysis. Here, my goal was to analyse published RNA-seq data sets to look for novel modifiers. I obtained RNA-seq data sets from laser capture microdissection (LCM) motor neurons from both SMNΔ7 and Smn²ᴮ/⁻ mice, from a range of resistant and vulnerable populations. I reanalysed and compared these data sets and identified a number of exciting potential modifiers. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), seemed to be an exciting potential neuroprotective modifier. PACAP has been shown to be neuroprotective in an array of neurodegenerative diseases but has not yet been investigated in models of SMA. Here, I investigated whether increased PACAP levels could ameliorate neuronal pathology in a mouse model of SMA. Firstly, PACAP was administered IP to the Smn²ᴮ/⁻ mouse model, showing no significant effect on survival, motor performance and selective vulnerability. Therefore, subsequently PACAP was administered via AAV9 delivery in the Smn²ᴮ/⁻ mouse model, showing no significant effects on the neuropathology in the Smn²ᴮ/⁻ mouse model. Finally, since I have identified significant divergence in relative vulnerability in the neurons of the facial motor nucleus, I aimed to perform high spatial resolution transcriptional profiling of motor neurons in the SMNΔ7 mouse model with the ultimate goal of identifying transcriptional signatures of differential vulnerability. I therefore optimised and applied a novel method to perform RNA sequencing on single MNs isolated from the facial nucleus of the brainstem by LCM. A total of 1050 single MNs have been isolated from the facial nucleus from individual P2, P5 and P9 SMNΔ7 mice and respective controls. cDNA libraries were prepared and cDNA fragmented, uniquely barcoded and sent for initial quality control RNA-seq analysis. The analysis revealed promising quality control parameters of the sequenced samples and thereby confirmed a successful optimisation of this novel sequencing technique. Overall, these results provide an important insight into selective vulnerability patterns of different SMA mouse models and identified novel mechanisms and transcripts that have the potential to contribute to the protection of motor neurons in SMA mouse models.

https://hdl.handle.net/1842/41160

http://dx.doi.org/10.7488/era/3896

en

The University of Edinburgh

Comley, L.H., Kline, R.A., Thomson, A.K., Woschitz, V., Landeros, E.V., Osman, E.Y., Lorson, C.L., Murray, L.M., 2022. Motor Unit Recovery Following Smn Restoration in Mouse Models of Spinal Muscular Atrophy. Human Molecular Genetics ddac097. https://doi.org/10.1093/hmg/ddac097

Woschitz, V., Mei, I., Hedlund, E., Murray, L.M., 2022. Mouse models of SMA show divergent patterns of neuronal vulnerability and resilience. Skeletal Muscle 12, 22. https://doi.org/10.1186/s13395-022-00305-9

spinal muscular atrophy

SMA

neuromuscular junction

NMJ

mouse model

PACAP

motor neurons

Investigating modifiers that can regulate selective vulnerability in mouse models of spinal muscular atrophy

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy