Abstract
The neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characteristically heterogeneous in both genetic causality and cellular pathology. In ALS, the upper and lower motor neurons that mediate voluntary movement degenerate and die. The associated clinical phenotype presents as loss of control of movement, resulting in gradual paralysis, with a predicted survival time of only three to five years. Multiple genetic mutations have been associated with disease onset, and mutations in the gene ANXA11 were recently shown to cause a subset of ALS cases. Neuronal functions of the corresponding protein, Annexin A11, are not well defined, and the cellular phenotypes associated with ANXA11 ALS mutations are poorly understood.Utilisation of induced pluripotent derived stem cells (iPSCs) to generate disease relevant cell types enables the comparison of dysregulated cellular pathways in patient and heathy cells, however iPSC models of many ALS associated genes do not exist, including for ANXA11. To address this, iPSCs were generated from five ALS patients with previously identified non-synonymous single nucleotide polymorphisms in ANXA11, and five sex-matched control individuals. Novel iPSC lines were subject to quality control analysis to ensure that they were pluripotent in nature and had maintained genomic stability in the reprogramming process. Efficient differentiation of iPSCs into motor neurons was established, alongside optimisation of motor neuron-astrocyte co-culture to generate a disease relevant platform for subsequent analyses.
Initial characterisation of ANXA11 patient and control neuronal cultures included high-throughput protein localisation analysis of Annexin A11, as well as disease hallmark TDP-43 and phosphorylated TDP-43. This was completed in motor neuron monocultures and motor neuron-astrocyte co-cultures at two time points. Protein characteristics varied across culture conditions, however did not indicate any striking disease associated protein phenotype in ANXA11 patient motor neurons. Spontaneous calcium activity in motor neuron-astrocyte co-cultures was shown at two time points and indicated an increased calcium fluctuation propensity in some ANXA11 patient-derived lines, which was not mirrored by any change in electrophysiological readout. Neurite outgrowth analysis revealed comparable neurite characteristics in young motor neurons, however utilisation of microfluidic devices indicated reduced integrity of distal axons in ANXA11 ALS motor neurons.
Annexin A11 localises to stress granules and has a role in tethering RNA binding proteins (RBPs) to lysosomes to enable RBP transport along axons. To interrogate RNA dynamics in ANXA11 patient motor neurons, stress granule analysis was completed. Annexin A11 localised to G3BP1 positive stress granules in motor neurons, and reduced stress granule formation propensity was seen in ANXA11 R235Q motor neurons compared to control. Global axonal RNA transport velocity was assessed, revealing an increase in RNA transport velocity in patient motor neurons. A role of Annexin A11 in local translation was hypothesised due to its function in tethering RBPs to membrane bound vesicles, and the distal axon phenotype identified in microfluidic devices. Annexin A11 was shown to localise to focal points of translation in ribopuromycylation analysis, and Annexin A11 and the ALS associated protein FUS localised to overlapping sites of local translation in motor neurons. Using microfluidics to compartmentalise distal axons, Annexin A11 and FUS protein were assessed at sites of local translation in control and ANXA11 patient motor neurons. This highlighted increased Annexin A11 signal and decreased FUS signal at sites of local translation in patient lines, and a potential reduction in co-localisation.
Collectively these data implicate ANXA11 ALS mutations in a multilevel pathology, whereby dysregulation of overlapping cellular pathways collectively lead to motor neuron vulnerability. Many of the implicated pathways are associated with other genetic forms of ALS, highlighting overlapping cellular perturbations across the ALS spectrum.
| Date of Award | 1 May 2023 |
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| Original language | English |
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| Supervisor | Agnes Nishimura (Supervisor), Christopher Shaw (Supervisor), Bradley Smith (Supervisor) & Graham Cocks (Supervisor) |