An in-vitro Testing Platform to Assess Functional Effects of Distinct Tsc2 Mutations on Neuronal Morphology and mTOR Signaling
Abstract number :
2.46
Submission category :
2. Translational Research / 2D. Models
Year :
2023
Submission ID :
1347
Source :
www.aesnet.org
Presentation date :
12/3/2023 12:00:00 AM
Published date :
Authors :
Presenting Author: Aynara Wulsin, MD PhD – Cincinnati Children's Hospital
Amanda McGann, BSc – Medical Student, Neurology, Cincinnati Children's Hospital; Candi LaSarge, PhD – Anesthesia – Cincinnati Children's Hospital; Darcy Krueger, MD PhD – Professor, Neurology, Cincinnati Children's Hospital; Steve Danzer, PhD – Professor, Neurology, Cincinnati Children's; Christina Gross, PhD – Associate Professor, Neurology, Cincinnati Children's Hospital
Rationale: Tuberous Sclerosis Complex (TSC) is a genetic disorder characterized by the development of benign tumors, autism, and epilepsy. The disease is caused by mutations in Tsc1 or Tsc2 genes, which lead to altered cell signal transduction, most prominently hyperactivation of the mechanistic target of rapamycin (mTOR) pathway. The phenotypic presentation can differ widely between patients; however, surprisingly, the biological consequences (and thus the contribution to disease severity) of the primary mutations in Tsc1 or Tsc2 are unknown. Here, we report a novel in-vitro testing platform that allows studying distinct human Tsc2 mutations to identify specific genotype-phenotype relationships that can help predict disease course.
Methods: We developed a lentiviral polycistronic vector to simultaneously delete the murine Tsc2 allele in neurons and express human TSC2 (hTsc2) with or without disease-associated mutations. The vector contains Cre recombinase and an N-terminally Flag-tagged hTsc2 allele under the control of the human synapsin promoter, ensuring that the human allele (wild type or mutant) will always replace the endogenous mouse allele in the same neuron. We tested the functionality of the vector and the effects of the gene replacement strategy using low-density primary cortical neuronal cultures generated from male and female mouse embryos carrying two floxed Tsc2 alleles on a C57BL/6J background. At seven days in vitro, embryonic cultures were transduced with Cre-only virus, Cre-hTsc2 (wildtype), or left untreated. After 21 days, we performed qRT-PCR, western blot, mTOR signaling pathway Luminex assay, and immunohistochemistry analysis to assess the functionality of the gene replacement strategy. Experiments were repeated four times using four different pregnant dams.
Results: QRT-PCR analyses using Cre-, hTsc2- and flag-specific primers verified that lentiviral transduction led to transgene expression as expected. Western blot analyses further confirmed murine Tsc2 deletion and re-expression of hTsc2. Like human data, fluorescence immunocytochemistry showed that neuronal cultures from the Tsc2 knockout (KO) group (Tsc2flox/flox neurons transduced with hSYN-Cre lentivirus) had increased phosphorylation of the mTOR downstream target S6 and enlarged soma size phenotypes (1-way ANOVA p< 0.001 n=6). The gene replacement vector co-expressing Cre and hTsc2 normalized phospho-S6 levels and soma size (1-way ANOVA p< 0.001 n=6) to those seen in wild-type controls. In pilot studies using Luminex assay technology, we confirmed that enhanced mTOR signaling was also restored in the hTsc2-replacement group compared with Tsc2 KO.
Conclusions: We have established a scalable platform to characterize Tsc2 mutations. Our model replicates key phenotypes seen in patient tissue samples and other Tsc2 mutation models, such as enlarged neuronal soma size and altered signal transduction, that can be rescued by gene replacement. In the future, this testing platform will allow us to rapidly establish genotype-phenotype relationships for patient-specific Tsc2 mutations, making a personalized medicine approach for TSC feasible.
Funding: RISE Award, CGRP Grant
Translational Research