Minute amounts of hamartin wildtype rescue the emergence of tuber-like lesions in conditional Tsc1 ablated mice

• Focal cortical Tsc1 loss (at > E14) results in tuber-like lesions in adult mice.
• Expression of disease-associated TSC1 mutations results in a Tsc1-KO phenotype.
• Minimal amounts of hamartin are sufficient to rescue the Tsc1-knockout phenotype.
• We propose silencing of the second TSC1 allele as pathomechanism of cortical tubers.

Tuberous sclerosis (TSC) is a phacomatosis associated with highly differentiated malformations including tubers in the brain. Those are composed of large dysplastic neurons and ‘giant cells’. Cortical tubers are frequent causes of chronic seizures and resemble neuropathologically focal cortical dysplasias (FCD) type IIb. Patients with FCDIIb, however, lack additional stigmata of TSC. Mutations and allelic variants of the TSC1 gene have been observed in patients with tubers as well as FCDIIb. Those include hamartinR692X and hamartinR786X, stop mutants frequent in TSC patients and hamartinH732Y frequent in FCDIIb. Expression of these variants in cell culture led to aberrant distribution of corresponding proteins. We here scrutinized morphological and structural effects of these TSC1 variants by intraventricular in utero electroporation (IUE), genetically mimicking the discrete focal character and a somatic postzygotic mosaicism of the lesion, focusing on the gene dosage required for tuber-like lesions to emerge in Tsc1flox/flox mice. Expression of only hamartinR692X as well as hamartinR786X led to a 2-fold enlargement of neurons with high pS6 immunoreactivity, stressing their in vivo pathogenic potential. Co-electroporation of the different aberrant alleles and varying amounts of wildtype TSC1 surprisingly revealed already minimal amounts of functional hamartin to be sufficient for phenotype rescue. This result strongly calls for further studies to unravel new mechanisms for substantial silencing of the second allele in cortical tubers, as proposed by Knudson's ‘2-hit hypothesis’. The rescuing effects may provide a promising basis for gene therapies aiming at reconstituting hamartin expression in tubers.