Authors :
Presenting Author: Victoria Riley, PhD – Cincinnati Children's Hospital Medical Center
Lilian Jerow, BS – Cincinnati Children's Hospital Medical Center
Mary Dusing, BS – Cincinnati Children's Hospital Medical Center
Candi LaSarge, PhD – Cincinnati Children's Hospital Medical Center
Steve Danzer, PhD – Cincinnati Children's Hospital Medical Center
Rationale:
Tuberous Sclerosis Complex (TSC) is an mTORopathy that is characterized by the formation of dysplastic cortical lesions. These lesions are believed to underlie epilepsy, affecting 90% of TSC patients. The major phenotypes of TSC, including dysplastic lesions, are often caused by “second hit” mutations in Tsc1 or Tsc2 wherein germline loss of one allele is followed by a second somatic mutation or loss-of-heterozygosity affecting the second allele, creating focal knockout (KO) lesions surrounded by heterozygous cells. Whether heterozygous tissues alter the pathogenicity of focal KO lesions is not known. To explore this question, we created a mouse model to produce focal loss of Tsc2 among excitatory neurons on either Tsc2 wildtype or heterozygous backgrounds.
Methods:
Tsc2fl/fl mice, in which loxP sites flank exons 2-4 of the Tsc2 gene, were crossed to a CRE recombinase (CRE) deleter line to generate Tsc2fl/- mice. When bred, mice can be produced that mimic TSC pathogenesis, wherein mice lose one copy of Tsc2 early in development (in this case in the germline) while the second allele can be deleted later in life using CRE. For the present study, mice were generated with a germline heterozygous mutation in Tsc2 with either a wildtype (Tsc2wt/-) or CRE-inducible second allele (Tsc2fl/-). To further mimic TSC, Tsc2fl/- mice were injected intracortically at postnatal day two with AAV9-CaMKII-CRE-mCherry (Tsc2AAV9) to generate focal lesions with total Tsc2 inactivation in both alleles among infected cortical excitatory neurons. Mice were monitored by video-EEG to assess seizure frequency and severity. Results:
Tsc2wt/- mice were healthy and did not develop seizures when regions with total Tsc2 loss were not present. When injected with AAV-CRE, Tsc2 inactivation in focal areas of the cortex resulted in lesions of variable size (1.178 ± 0.343 mm3) and seizures in 50% of mice (N = 12). Seizure onset was on average 4.14 ± 1.65 days after EEG implantation (N = 5) with a frequency of 4.38 ± 0.77 seizures per day (N = 6). Seizures had an average duration of 36.74 ± 3.89 seconds (N = 4) with a Racine score of 4.45 ± 0.19 (N = 4).Conclusions:
We have developed a model of TSC that recapitulates the genetic condition of patients. This model is useful as it mimics the exon deletion strategy used commonly in previous TSC research and enables the loss of identical mutations at different time points in development. Importantly, our mouse model does not have seizures in the absence of focal regions of Tsc2 loss, but does have seizures when knockout lesions are present, affirming that seizure activity requires full Tsc2 inactivation. In the future, studies will include the examination of focal cortical Tsc2 null lesions on different genetic backgrounds, such as germline heterozygous and wildtype backgrounds. How and if seizures differ depending on genetic background and the influence that the heterozygosity of Tsc2 may have on TSC pathogenesis remains to be determined.Funding:
This work was funded by RO1NS065020 and RO1NS062806.