G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Drug repurposing for Dravet syndrome in scn1Lab-/- mutant zebrafish.

Dravet syndrome (DS) is a severe genetic epileptic encephalopathy with onset during the first year of life. Zebrafish models recapitulating human diseases are often used as drug discovery platforms, but also for drug repurposing testing. It was recently shown that pharmacological modulation of three serotonergic (5-HT) receptors (5-HT1D , 5-HT2C , 5-HT2A ) exerts antiseizure effects in a zebrafish scn1Lab-/- mutant model of DS. Using the zebrafish DS model, our aim was to examine the possibility of repurposing efavirenz (EFA), lisuride (LIS), and rizatriptan (RIZA), marketed medicines with a 5-HT on- or off-target profile, as antiepileptic drugs for DS. To examine whether these compounds have a broader antiseizure profile, they were tested in pentylenetetrazol and ethyl ketopentenoate (EKP) zebrafish models. Pharmacological effects were assessed by locomotor behavior, local field potential brain recordings, and bioluminescence. EFA was active in all models, whereas LIS was selectively active in the zebrafish DS model. Mainly, a poor response was observed to RIZA. Taken together, our preclinical results show that LIS could be a potential candidate for DS treatment. EFA was also active in the EKP model, characterized by a high level of treatment resistance, and hence these data are potentially important for future treatment of drug-resistant epilepsy.

mTOR-related neuropathology in mutant tsc2 zebrafish: Phenotypic, transcriptomic and pharmacological analysis.

Tuberous sclerosis complex (TSC) is a rare, genetic disease caused by loss-of-function mutations in either TSC1 or TSC2. Patients with TSC are neurologically characterized by the presence of abnormal brain structure, intractable epilepsy and TSC-associated neuropsychiatric disorders. Given the lack of effective long-term treatments for TSC, there is a need to gain greater insight into TSC-related pathophysiology and to identify and develop new treatments. In this work we show that homozygous tsc2-/- mutant zebrafish larvae, but not tsc2+/- and WT larvae, display enlarged brains, reduced locomotor behavior and epileptiform discharges at 7dpf. In addition, we pharmacologically validated the TSC model by demonstrating the dramatic rescue effect of pericardially injected rapamycin, a well-known mTOR inhibitor, on selected behavioral read-outs and at the molecular level. By means of trancriptome profiling we also acquired more insight into the neuropathology of TSC, and as a result were able to highlight possible new treatment targets. The gene expression profiles of WT and tsc2+/- larvae revealed 117 differentially expressed genes (DEGs), while between WT and tsc2-/- larvae and tsc2+/- and tsc2-/- larvae there were 1414 and 1079 DEGs, respectively. Pathway enrichment analysis from the WT and tsc2-/- DEGs, identified 14 enriched pathways from the up-regulated genes and 6 enriched pathways from the down-regulated genes. Moreover, genes related to inflammation and immune response were up-regulated in the heads of tsc2-/- larvae, in line with the findings in human brain tissue where inflammatory and immune responses appear to be major hallmarks of TSC. Taken together, our phenotypic, transcriptomic and pharmacological analysis identified the tsc2-/- zebrafish as a preclinical model that mirrors well aspects of the human condition and delineated relevant TSC-related biological pathways. The model may be of value for future TSC-related drug discovery and development programs.

Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in the TSC1 or TSC2 genes and is characterized by widespread tumour growth, intractable epilepsy, cognitive deficits and autistic behaviour. CBD has been reported to decrease seizures and inhibit tumour cell progression, therefore we sought to determine the influence of CBD on TSC pathology in zebrafish carrying a nonsense mutation in the tsc2 gene. CBD treatment from 6 to 7 days post-fertilization (dpf) induced significant anxiolytic actions without causing sedation. Furthermore, CBD treatment from 3 dpf had no impact on tsc2-/- larvae motility nor their survival. CBD treatment did, however, reduce the number of phosphorylated rpS6 positive cells, and their cross-sectional cell size. This suggests a CBD mediated suppression of mechanistic target of rapamycin (mTOR) activity in the tsc2-/- larval brain. Taken together, these data suggest that CBD selectively modulates levels of phosphorylated rpS6 in the brain and additionally provides an anxiolytic effect. This is pertinent given the alterations in mTOR signalling in experimental models of TSC. Additional work is necessary to identify upstream signal modulation and to further justify the use of CBD as a possible therapeutic strategy to manage TSC.

Gain-of-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy.

OBJECTIVE: Voltage-gated sodium channel (Nav)-encoding genes are among early-onset epileptic encephalopathies (EOEE) targets, suggesting that other genes encoding Nav-binding proteins, such as fibroblast growth factor homologous factors (FHFs), may also play roles in these disorders. METHODS: To identify additional genes for EOEE, we performed whole-exome sequencing in a family quintet with 2 siblings with a lethal disease characterized by EOEE and cerebellar atrophy. The pathogenic nature and functional consequences of the identified sequence alteration were determined by electrophysiologic studies in vitro and in vivo. RESULTS: A de novo heterozygous missense mutation was identified in the FHF1 gene (FHF1AR114H, FHF1BR52H) in the 2 affected siblings. The mutant FHF1 proteins had a strong gain-of-function phenotype in transfected Neuro2A cells, enhancing the depolarizing shifts in Nav1.6 voltage-dependent fast inactivation, predicting increased neuronal excitability. Surprisingly, the gain-of-function effect is predicted to result from weaker interaction of mutant FHF1 with the Nav cytoplasmic tail. Transgenic overexpression of mutant FHF1B in zebrafish larvae enhanced epileptiform discharges, demonstrating the epileptic potential of this FHF1 mutation in the affected children. CONCLUSIONS: Our data demonstrate that gain-of-function FHF mutations can cause neurologic disorder, and expand the repertoire of genetic causes (FHF1) and mechanisms (altered Nav gating) underlying EOEE and cerebellar atrophy.