Hyperactivation of mTORC1 in a double hit mutant zebrafish model of tuberous sclerosis complex causes increased seizure susceptibility and neurodevelopmental abnormalities

Abstract

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by pathogenic variants in TSC1 and TSC2 genes. TSC patients present with seizures and brain abnormalities such as tubers and subependymal giant cells astrocytoma (SEGA). Despite common molecular and clinical features, the severity of the disease varies greatly, even intrafamilially. The second hit hypothesis suggests that an additional, inactivating mutation in the remaining functional allele causes a more severe phenotype and therefore explains the phenotypic variability. Recently, second hit mutations have been detected frequently in mTORopathies. To investigate the pathophysiological effects of second hit mutations, several mouse models have been developed. Here, we opted for a double mutant zebrafish model that carries a LOF mutation both in the tsc2 and the depdc5 gene. To the best of our knowledge, this is the first time a second-hit model has been studied in zebrafish. Significantly, the DEP domain-containing protein 5 (DEPDC5) gene has an important role in the regulation of mTORC1, and the combination of a germline TSC2 and somatic DEPDC5 mutation has been described in a TSC patient with intractable epilepsy. Our depdc5−/−x tsc2−/− double mutant zebrafish line displayed greatly increased levels of mammalian target of rapamycin (mTORC1) activity, augmented seizure susceptibility, and early lethality which could be rescued by rapamycin. Histological analysis of the brain revealed ventricular dilatation in the tsc2 and double homozygotes. RNA-sequencing showed a linear relation between the number of differentially expressed genes (DEGs) and the degree of mTORC1 hyperactivity. Enrichment analysis of their transcriptomes revealed that many genes associated with neurological developmental processes were downregulated and mitochondrial genes were upregulated. In particular, the transcriptome of human SEGA lesions overlapped strongly with the double homozygous zebrafish larvae. The data highlight the clinical relevance of the depdc5−/− x tsc2−/− double mutant zebrafish larvae that showed a more severe phenotype compared to the single mutants. Finally, analysis of gene-drug interactions identified interesting pharmacological targets for SEGA, underscoring the value of our small zebrafish vertebrate model for future drug discovery efforts.

Developing Novel Experimental Models of m-TORopathic Epilepsy and Related Neuropathologies: Translational Insights from Zebrafish

“…These findings not only reinforce the role of mTOR in zebrafish models of epilepsy, but also link it to aberrant GABA signaling. In line with this, mTORC1 hyperactivity, early lethality and ventricular dilatation in the DEPDC5 −/− tsc2 −/− double-mutant zebrafish also occurs with augmented seizure susceptibility that is corrected by rapamycin [ 116 ].…”

A Case Report of Pediatric Patient with Tuberous Sclerosis and Radiologically Isolated Syndrome

“…This complex is a crucial controller of protein and lipid biosynthesis and cell-cycle progression associated with the growth factor, and plays a key role in neurodevelopment [3,4]. Under ordinary circumstances, hamartin and tuberin are activated via biosynthetic processes mediated by the mTOR complex 1 (mTORC1), which includes mTOR, raptor (protein associated with mTOR regulation), mLST8, and PRAS40 (prolinerich Akt substrate 40) [5]. Therefore, TSC1 or TSC2 mutations give rise to hyperactivation of the mTOR pathway, resulting in a downstream kinase signaling cascade that can lead to abnormalities in numerous cellular processes, including cell cycle progression, transcription, translation, and metabolic control [5].…”

“…Under ordinary circumstances, hamartin and tuberin are activated via biosynthetic processes mediated by the mTOR complex 1 (mTORC1), which includes mTOR, raptor (protein associated with mTOR regulation), mLST8, and PRAS40 (prolinerich Akt substrate 40) [5]. Therefore, TSC1 or TSC2 mutations give rise to hyperactivation of the mTOR pathway, resulting in a downstream kinase signaling cascade that can lead to abnormalities in numerous cellular processes, including cell cycle progression, transcription, translation, and metabolic control [5]. mTORC1 signaling has been demonstrated to be early activated during brain development, and pathological neural networks are therefore already present in the prenatal period.…”