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.

TrkB hyperactivity contributes to brain dysconnectivity, epileptogenesis, and anxiety in zebrafish model of Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is a rare genetic disease that manifests with early symptoms, including cortical malformations, childhood epilepsy, and TSC-associated neuropsychiatric disorders (TANDs). Cortical malformations arise during embryonic development and have been linked to childhood epilepsy before, but the underlying mechanisms of this relationship remain insufficiently understood. Zebrafish have emerged as a convenient model to study elementary neurodevelopment; however, without in-depth functional analysis, the Tsc2-deficient zebrafish line cannot be used for studies of TANDs or new drug screening. In this study, we found that the lack of Tsc2 in zebrafish resulted in heterotopias and hyperactivation of the mTorC1 pathway in pallial regions, which are homologous to the mammalian cortex. We observed commissural thinning that was responsible for brain dysconnectivity, recapitulating TSC pathology in human patients. The lack of Tsc2 also delayed axonal development and caused aberrant tract fasciculation, corresponding to the abnormal expression of genes involved in axon navigation. The mutants underwent epileptogenesis that resulted in nonmotor seizures and exhibited increased anxiety-like behavior. We further mapped discrete parameters of locomotor activity to epilepsy-like and anxiety-like behaviors, which were rescued by reducing tyrosine receptor kinase B (TrkB) signaling. Moreover, in contrast to treatment with vigabatrin and rapamycin, TrkB inhibition rescued brain dysconnectivity and anxiety-like behavior. These data reveal that commissural thinning results in the aberrant regulation of anxiety, providing a mechanistic link between brain anatomy and human TANDs. Our findings also implicate TrkB signaling in the complex pathology of TSC and reveal a therapeutic target.

Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23.

Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.

Tuberous sclerosis complex: From molecular biology to novel therapeutic approaches.

Tuberous sclerosis complex (TSC) is a rare multi-system disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, including the brain. TSC patients often suffer from epilepsy, mental retardation, and autism spectrum disorder (ASD). Therefore, TSC serves as a model of epilepsy, ASD, and tumorigenesis. TSC is caused by the lack of functional Tsc1-Tsc2 complex, which serves as a major cellular inhibitor of mammalian Target of Rapamycin Complex 1 (mTORC1). mTORC1 is a kinase controlling most of anabolic processes in eukaryotic cells. Consequently, mTORC1 inhibitors, such as rapamycin, serve as experimental or already approved drugs for several TSC symptoms. However, rapalogs, although quite effective, need to be administered chronically and likely for a lifetime, since therapy discontinuation results in tumor regrowth and epilepsy recurrence. Recent studies revealed that metabolism and excitability (in the case of neurons) of cells lacking Tsc1-Tsc2 complex are changed, and these features may potentially be used to treat some of TSC symptoms. In this review, we first provide basic facts about TSC and its molecular background, to next discuss the newest findings in TSC cell biology that can be used to improve existing therapies of TSC and other diseases linked to mTORC1 hyperactivation. © 2016 IUBMB Life, 68(12):955-962, 2016.

Hyperactive mTORC1 disrupts habenula function and light preference in zebrafish model of Tuberous sclerosis complex.

Mechanistic target of rapamycin complex 1 (mTORC1) is an integration hub for extracellular and intracellular signals necessary for brain development. Hyperactive mTORC1 is found in autism spectrum disorder (ASD) characterized by atypical reactivity to sensory stimuli, among other symptoms. In Tuberous sclerosis complex (TSC) inactivating mutations in the TSC1 or TSC2 genes result in hyperactivation of the mTORC1 pathway and ASD. Here, we show that lack of light preference of the TSC zebrafish model, tsc2 vu242/vu242 is caused by aberrant processing of light stimuli in the left dorsal habenula and tsc2 vu242/vu242 fish have impaired function of the left dorsal habenula, in which neurons exhibited higher activity and lacked habituation to the light stimuli. These characteristics were rescued by rapamycin. We thus discovered that hyperactive mTorC1 caused aberrant habenula function resulting in lack of light preference. Our results suggest that mTORC1 hyperactivity contributes to atypical reactivity to sensory stimuli in ASD.

Adaptor Complex 2 Controls Dendrite Morphology via mTOR-Dependent Expression of GluA2.

The formation of dendritic arbors in neurons is a highly regulated process. Among the regulators of dendritogenesis are numerous membrane proteins that are eventually internalized via clathrin-mediated endocytosis. AP2 is an adaptor complex that is responsible for recruiting endocytic machinery to internalized cargo. Its direct involvement in dendritogenesis in mammalian neurons has not yet been tested. We found that the knockdown of AP2b1 (ß2-adaptin), an AP2 subunit, reduced the number of dendrites in developing rat hippocampal neurons and decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels by inhibiting mechanistic/mammalian target of rapamycin (mTOR). The dendritic tree abruption that was caused by AP2b1 knockdown was rescued by the overexpression of GluA2 or restoration of the activity of the mTOR effector p70S6 kinase (S6K1). Altogether, this work provides evidence that the AP2 adaptor complex is needed for the dendritogenesis of mammalian neurons and reveals that mTOR-dependent GluA2 biosynthesis contributes to this process.

Molecular neurobiology of mTOR.

Mammalian/mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that controls several important aspects of mammalian cell function. mTOR activity is modulated by various intra- and extracellular factors; in turn, mTOR changes rates of translation, transcription, protein degradation, cell signaling, metabolism, and cytoskeleton dynamics. mTOR has been repeatedly shown to participate in neuronal development and the proper functioning of mature neurons. Changes in mTOR activity are often observed in nervous system diseases, including genetic diseases (e.g., tuberous sclerosis complex, Pten-related syndromes, neurofibromatosis, and Fragile X syndrome), epilepsy, brain tumors, and neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, and Huntington's disease). Neuroscientists only recently began deciphering the molecular processes that are downstream of mTOR that participate in proper function of the nervous system. As a result, we are gaining knowledge about the ways in which aberrant changes in mTOR activity lead to various nervous system diseases. In this review, we provide a comprehensive view of mTOR in the nervous system, with a special focus on the neuronal functions of mTOR (e.g., control of translation, transcription, and autophagy) that likely underlie the contribution of mTOR to nervous system diseases.