Neuronal CTGF/CCN2 negatively regulates myelination in a mouse model of tuberous sclerosis complex.

Disruption of myelination during development has been implicated in a range of neurodevelopmental disorders including tuberous sclerosis complex (TSC). TSC patients with autism display impairments in white matter integrity. Similarly, mice lacking neuronal Tsc1 have a hypomyelination phenotype. However, the mechanisms that underlie these phenotypes remain unknown. In this study, we demonstrate that neuronal TSC1/2 orchestrates a program of oligodendrocyte maturation through the regulated secretion of connective tissue growth factor (CTGF). We characterize oligodendrocyte maturation both in vitro and in vivo. We find that neuron-specific Tsc1 deletion results in an increase in CTGF secretion that non-cell autonomously stunts oligodendrocyte development and decreases the total number of oligodendrocytes. Genetic deletion of CTGF from neurons, in turn, mitigates the TSC-dependent hypomyelination phenotype. These results show that the mechanistic target of rapamycin (mTOR) pathway in neurons regulates CTGF production and secretion, revealing a paracrine mechanism by which neuronal signaling regulates oligodendrocyte maturation and myelination in TSC. This study highlights the role of mTOR-dependent signaling between neuronal and nonneuronal cells in the regulation of myelin and identifies an additional therapeutic avenue for this disease.

A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS.

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

TSC1/TSC2 signaling in the CNS.

Over the past several years, the study of a hereditary tumor syndrome, tuberous sclerosis complex (TSC), has shed light on the regulation of cellular proliferation and growth. TSC is an autosomal dominant disorder that is due to inactivating mutations in TSC1 or TSC2 and characterized by benign tumors (hamartomas) involving multiple organ systems. The TSC1/2 complex has been found to play a crucial role in an evolutionarily-conserved signaling pathway that regulates cell growth: the mTORC1 pathway. This pathway promotes anabolic processes and inhibits catabolic processes in response to extracellular and intracellular factors. Findings in cancer biology have reinforced the critical role for TSC1/2 in cell growth and proliferation. In contrast to cancer cells, in the CNS, the TSC1/2 complex not only regulates cell growth/proliferation, but also orchestrates an intricate and finely tuned system that has distinctive roles under different conditions, depending on cell type, stage of development, and subcellular localization. Overall, TSC1/2 signaling in the CNS, via its multi-faceted roles, contributes to proper neural connectivity. Here, we will review the TSC signaling in the CNS.

Tsc2-Rheb signaling regulates EphA-mediated axon guidance.

Tuberous sclerosis complex is a disease caused by mutations in the TSC1 or TSC2 genes, which encode a protein complex that inhibits mTOR kinase signaling by inactivating the Rheb GTPase. Activation of mTOR promotes the formation of benign tumors in various organs and the mechanisms underlying the neurological symptoms of the disease remain largely unknown. We found that Tsc2 haploinsufficiency in mice caused aberrant retinogeniculate projections that suggest defects in EphA receptor-dependent axon guidance. We also found that EphA receptor activation by ephrin-A ligands in neurons led to inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity and decreased inhibition of Tsc2 by ERK1/2. Thus, ephrin stimulation inactivates the mTOR pathway by enhancing Tsc2 activity. Furthermore, Tsc2 deficiency and hyperactive Rheb constitutively activated mTOR and inhibited ephrin-induced growth cone collapse. Our results indicate that TSC2-Rheb-mTOR signaling cooperates with the ephrin-Eph receptor system to control axon guidance in the visual system.

Sodium currents in mesencephalic trigeminal neurons from Nav1.6 null mice.

Previous studies using pharmacological methods suggest that subthreshold sodium currents are critical for rhythmical burst generation in mesencephalic trigeminal neurons (Mes V). In this study, we characterized transient (I(NaT)), persistent (I(N)(aP)), and resurgent (I(res)) sodium currents in Na(v)1.6-null mice (med mouse, Na(v)1.6(-/-)) lacking expression of the sodium channel gene Scn8a. We found that peak transient, persistent, and resurgent sodium currents from med (Na(v)1.6(-/-)) mice were reduced by 18, 39, and 76% relative to their wild-type (Na(v)1.6(+/+)) littermates, respectively. Current clamp recordings indicated that, in response to sinusoidal constant amplitude current (ZAP function), all neurons exhibited membrane resonance. However, Mes V neurons from med mice had reduced peak amplitudes in the impedance-frequency relationship (resonant Q-value) and attenuated subthreshold oscillations despite the similar passive membrane properties compared with wild-type littermates. The spike frequency-current relationship exhibited reduced instantaneous discharge frequencies and spike block at low stimulus currents and seldom showed maintained spike discharge throughout the stimulus in the majority of med neurons compared with wild-type neurons. Importantly, med neurons never exhibited maintained stimulus-induced rhythmical burst discharge unlike those of wild-type littermates. The data showed that subthreshold sodium currents are critical determinants of Mes V electrogenesis and burst generation and suggest a role for resurgent sodium currents in control of spike discharge.

Participation of sodium currents in burst generation and control of membrane excitability in mesencephalic trigeminal neurons.

Subthreshold sodium currents are important in sculpting neuronal discharge and have been implicated in production and/or maintenance of subthreshold membrane oscillations and burst generation in mesencephalic trigeminal neurons (Mes V). Moreover, recent data suggest that, in some CNS neurons, resurgent sodium currents contribute to production of high-frequency burst discharge. In the present study, we sought to determine more directly the participation of these currents during Mes V electrogenesis using the action potential-clamp method. In postnatal day 8-14 rats, the whole-cell patch-clamp method was used to record sodium currents by subtraction in response to application of TTX in voltage-clamp mode using the action potential waveform as the command protocol. We found that TTX-sensitive sodium current is the main inward current flowing during the interspike interval, compared with the h-current (Ih) and calcium currents. Furthermore, in addition to the transient sodium current that flows during the upstroke of action potential, we show that resurgent sodium current flows at the peak of afterhyperpolarization and persistent sodium current flows in the middle of the interspike interval to drive high-frequency firing. Additionally, transient, resurgent, and persistent sodium current components showed voltage- and time-dependent slow inactivation, suggesting that slow inactivation of these currents can contribute to burst termination. The data suggest an important role for these components of the sodium current in Mes V neuron electrogenesis.