Authors :
Presenting Author: Lilian Jerow, BS – University of Cincinnati College of Medicine
Candi LaSarge, PhD – Cincinnati Children's Hospital Medical Center
Carlie McCoy, BS – Cincinnati Children's Hospital Medical Center
Mary Dusing, BS – Cincinnati Children's Hospital Medical Center
Steve Danzer, PhD – Cincinnati Children's Hospital Medical Center
Rationale:
Tuberous sclerosis complex (TSC) is a neurocutaneous disorder affecting nearly 1 in 6,000 individuals, with epilepsy being the most common neurological symptom. TSC occurs from mutations to either the TSC1 or TSC2 genes, resulting in hyperactivation of the mTOR pathway. Previously, TSC was believed to be a disorder occurring from mutations within radial glial progenitor cells producing excitatory neurons. However, recent research has shown the involvement of interneurons in some patients. Whether interneuron involvement alters disease phenotype is unknown. Previously, we developed a model of TSC with a focal deletion of Tsc2 from excitatory neurons. Here we generate a new model deleting Tsc2 from excitatory neurons and somatostatin (SST)-expressing interneurons to assess the role of interneurons in TSC.
Methods:
To concurrently delete Tsc2 from excitatory neurons and SST interneurons, a triple transgenic (SST-FlpO, tdTomato (tdT) reporter, Tsc2fl/fl) dual viral injection approach was developed. To target excitatory cells, AAV9-CamKII-Cre-EGFP was injected into cortex to initiate cre-recombinase-mediated deletion of Tsc2 from CamKII-expressing excitatory neurons. To target SST interneurons, Flp recombinase was expressed in SST neurons using SST-FlpO mice, leading to Tsc2 deletion via Flp-dependent AAV9-CAG-Frt-Cre/HA infection. With this approach, excitatory cells express GFP from the AAV and tdT from the reporter line, while SST neurons are tagged with hemagglutinin (HA) from the AAV and tdT. AAV was infused on postnatal day 2 (P2) via two injections (100nl each) of mixed AAV9-CAG-Frt-Cre/HA + AAV9-CaMKII-Cre-EGFP into frontal cortex. SST-FlpO+/-, Tdt+/-, Tsc2wt/wt mice served as controls. At 8-12 weeks tissue was collected and processed for histology.
Results:
Dual AAV injection at equal titers (2.25x109 gc/µl) resulted in a high infection rate of excitatory cells and minimal infection of somatostatin interneurons. Variations in viral titer and injection volume were tested to optimize labeling of both cell types. Best results were obtained by increasing the viral titer of AAV9-CAG-Frt-Cre/HA to 3.5x109 gc/µl while lowering the titer of AAV9-CamKII-Cre-EGFP to 1.125x109 gc/µl. This combination produced high infectivity of inhibitory neurons, labeling 79.5-90.5% of SST cells in the injected region. Moreover, within the injected region, SST interneurons comprised ~16.5-25.83% of all tdT+ cells, the remainder being excitatory neurons (n = 3). Histological analyses confirmed Tsc2 deletion and increased pS6 expression in somatostatin interneurons.
Conclusions:
This study demonstrates the feasibility of generating focal cortical Tsc2 KO lesions that contain both excitatory and inhibitory neurons. Findings further demonstrate that Tsc2 KO interneurons exhibited elevated pS6 immunoreactivity, indicative of mTOR hyperactivation. The model will help determine whether inclusion of SST interneurons in cortical malformations in mTORopathies impacts pathological, seizure or behavioral phenotypes.
Funding:
R01NS065020 and R01NS062806