Objective: To investigate phenotypic differences between iPSC-differentiated cortical neurons derived from patients with TOR1A dystonia compared to healthy controls.

Background: Primary generalised dystonia is most commonly caused by autosomal dominant TOR1A gene mutation. How TOR1A mutation disrupts neuronal function is unknown. Previous cellular and murine models of TOR1A dystonia describe changes in nuclear envelope structure, with accumulation of ubiquitin and nuclear pore complex (NPC) proteins within nuclear envelope buds [1]. Alterations in corticostriatal synaptic plasticity [2] and changes in striatal synaptic levels of numerous neurotransmitter receptors and transporters [3, 4,] have been reported.

Method: We developed induced pluripotent stem cell (iPSC) lines from the fibroblasts of patients with TOR1A dystonia and differentiated them into glutamatergic cortical neurons according to established protocols [5]. Three TOR1A patient-derived iPSC lines and three controls underwent fixation and immunostaining at day 60. The following assays were performed:

1. TubulinβIII stain to assess neuritic outgrowth and lamininB stain to examine nuclear envelope structure.

2. MAP2 stain to assess for changes in dendritic arborization and Synaptophysin stain as a marker of synaptic development.

3. Ubiquitin (linkage-specific K48) antibody with NPC (mAb414) antibody staining to assess for the presence of nuclear envelope ubiquitin inclusions and nuclear pore complex (NPC) clustering

Results: Both the cases and controls demonstrated positive nuclear ubiquitin-K48 staining, without differences in NPC morphology. There were no changes in nuclear envelope structure. However, there was greater neurite branching and greater dendritic synaptophysin staining in patient-derived neurons compared to controls.

Conclusion: TOR1A patient-derived cortical neurons do not demonstrate the nuclear envelope abnormalities observed in iPSC-derived ventral motor neurons [6], suggesting differential effects of TorsinA dysfunction among different neuronal types. Preliminary immunostaining at Day60 suggested more complex neuritic architecture and greater dendritic synaptophysin staining in patient-derived neurons compared to controls which may indicate enhanced neuronal maturation at this early timepoint. We currently are undertaking western blot assays and immunocytochemistry at later developmental timepoints to investigate these trends.

References: [1] Pappas S et al., (2018). TorsinA dysfunction causes persistent neuronal nuclear pore defects. Hum Mol Genet 27; 407-420.
[2] Martella G et al., (2008). Impairment of bidirectional synaptic plasticity in the striatum of a mouse model of DYT1 dystonia: role of endogenous acetylcholine. Brain 132: 2336–2349.
[3] Maltese M et al., (2018). Early structural and functional plasticity alterations in a susceptibility period of DYT1 dystonia mouse striatum. eLife 7: e33331.
[4] Tassone A et al., (2021).Vesicular Acetylcholine Transporter Alters Cholinergic Tone and Synaptic Plasticity in DYT1 Dystonia. Mov Disord 36; 2768-2779.
[5] Shi Y et al., (2012). Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci 15; 477–486
[6] Ding B et al., (2021).Disease Modeling with Human Neurons Reveals LMNB1 Dysregulation Underlying DYT1 Dystonia. J Neurosci 41; 2024-2038
[7] Wrigley S et al., (2023). Exploring the effects of torsinA dysfunction in an iPSC-derived cortical neuronal model of DYT1-TOR1A dystonia (abstract). Samuel Belzberg 6th International Dystonia Symposium.

« Back to 2023 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/exploring-the-effects-of-torsina-dysfunction-in-an-ipsc-derived-neuronal-model-of-tor1a-dystonia/