Abstracts

Rationale: mTOR pathway mutations have been associated with autism, cognitive dysfunction and epilepsy. Interestingly, humans with mTOR pathway mutations often present with mosaic disruptions of gene function, producing lesions that range from focal cortical dysplasia to hemimegalencephaly. In animal models, loss of the mTOR pathway negative regulator PTEN from hippocampal dentate granule cells leads to neuronal hypertrophy, increased dendritic branching and aberrant basal dendrite formation. These mice are also predisposed to spontaneous seizures. Whether mTOR-mediated neuronal dysmorphogenesis is impacted by the number of affected cells, however, is not known. Secondary comorbidities in mTOR mutant mice could exacerbate dysmorphogenesis among mutant cells. Here, we tested the hypothesis that structural abnormalities among individual mTOR mutant neurons would be partly regulated by the number of surrounding mutant cells. Methods: To determine whether the percentage or “load” of PTEN knockout (KO) granule cells impacts the morphological development of these cells, 2-3 week old Gli1-CreER^T2 hemizygous; PTEN^flox/flox mice were treated with tamoxifen, yielding animals with PTEN deleted from 2-34% of granule cells (n=34 mice). Animals were sacrificed between 8-33 weeks-of-age, and acute hippocampal slices were prepared. A total of 48 individual PTEN KO cells were filled with biocytin to reveal their morphology, which was reconstructed using Neurolucida software. Morphological parameters were analyzed using multiple linear regression with age and %KO as independent variables. Results: Statistical modeling revealed that both age (p=0.001) and the percentage of KO cells (p=0.003) predicted the total length of the apical dendritic tree. The length of KO cell basal dendrites – abnormal process typically absent from healthy cells – was predicted by age (p=0.047) but not the percentage of KO cells (p=0.534). Conclusions: mTOR pathway mutations are linked to autism spectrum disorder, brain malformations and epilepsy. Mosaicism in these patients can produce widely varying numbers of mutant cells. Our findings indicate that the dendritic growth of individual KO cells is greater in animals with more KO cells, implying that disease severity in patients may reflect the combinatorial effects of the total size of the mutant cell population and the severity of abnormalities exhibited by individual mutant cells. Furthermore, mTOR mutant neurons may be functionally distinct among patients depending on whether there are a few or many mutant cells present. Funding: This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Awards R01NS065020 and R01NS062806; CLL, F32NS083239).