Investigating the role of Stromalin and its potential downstream targets on seizure behaviour in Drosophila

• Author / Creator

  Sai Dwijesha, Amoolya

• The cohesin complex is a highly conserved structure that plays an important role in sister chromatid cohesion, segregating chromosomes during cell division, gene regulation, and repairing double stranded DNA breaks. The cohesin complex is comprised of four core subunits: Stromalin (STAG1/2), Rad21, SMC1 and SMC3 as well as auxiliary proteins that aid the complex in its functions and maintenance. Mutations in the subunits and auxiliary proteins have been shown to result in a group of rare, multi-organ system-wide developmental disorder known as cohesinopathies that are characterized by behavioural, neurological, and growth abnormalities. Past studies on cohesinopathies have provided evidence for dysregulated gene expression and not chromosomal segregation as a pathological mechanism underlying the disease. One of the neurological symptoms in cohesinopathies is an increased rate of seizures, particularly seen in patients with mutations in STAG1/2 and SMC1 subunits. However, the mechanisms underlying this remain unknown.
  Recently, the Drosophila homologue of the STAG1/2 subunit, Stromalin, was found to affect learning and memory by constraining the synaptic vesicle pool sizes and synaptic communication in Drosophila dopamine neurons. As an imbalance in synaptic communication is typically associated with seizures, we hypothesized that the synaptic vesicle and synaptic communication increases upon Stromalin knockdown may be a potential mechanism underlying contributing to seizures in cohesinopathies. We performed a standard mechanical seizure induction assay on Drosophila expressing RNAi targeted against stromalin and SMC1 in the brain, and found these manipulations significantly increased seizure frequencies, consistent with observations in cohesinopathy patients. We then tested 5 gene candidates, Nep1, CG17698, Cox7c, Ttm2 and Su(z)12, that were identified to potentially regulate the cohesin complex’s effects on memory, for their ability to induce seizures. Knockdown of CG17698 and Cox7c in the brain using RNAi increased seizure frequencies in our animals, but not Nep1, CG17698, or Ttm2.
  We then investigated the role of synaptic vesicles in the increased seizure rates we were seeing in our flies. We decreased the numbers of synaptic vesicle numbers at the synapses by impairing unc104 function, which is known to traffic synaptic vesicles from the cell body to the synaptic terminal. We found that the pan-neuronal reduction of the synaptic vesicle trafficking protein, unc104 failed to reduce the seizure frequency in Stromalin knockdown flies, however the positive control did not show increased seizure frequency in this experiment. Thus, this interpretation may not be accurate. Finally, we also analyzed the intensities of a synaptic vesicle marker, Synaptotagmin:GFP using fluorescence microscopy. We found inconsistent changes in Synaptotagmin:GFP across the whole-brain in our flies and upon the pan-neuronal knockdown of unc104, suggesting that an association between changes in synaptic vesicle numbers and seizure phenotypes in our flies could not be confidently inferred from our data.
  All in all, through this project we have provided evidence for a potential Stromalin based Drosophila cohesinopathy model that has increased seizure frequencies, consistent with the increased seizure rates seen in patients with STAG1/2 and SMC1 mutations. Reduction of Stromalin and SMC1 may induce the seizure phenotype in flies by reducing transcription of CG17698 and Cox7c in neurons. Our findings suggest that certain neurological symptoms of cohesinopathies can be studied in Drosophila.

• Subjects / Keywords

  • Drosophila behaviour
  • Seizures
  • Stromalin
  • Cohesinopathies
  • Synaptic vesicle pool sizes

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-m69p-1719

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.