Tuberous sclerosis complex neuropathology requires glutamate-cysteine ligase

Introduction: Tuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy. Results: The results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients’ brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells. Conclusions: We describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0225-z) contains supplementary material, which is available to authorized users

Spatiotemporal characterization of mTOR kinase activity following kainic acid induced status epilepticus and analysis of rat brain response to chronic rapamycin treatment.

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain

Cell-type-specific translational control of spatial working memory by the cap-binding protein 4EHP

Abstract The consolidation of learned information into long-lasting memories requires the strengthening of synaptic connections through de novo protein synthesis. Translation initiation factors play a cardinal role in gating the production of new proteins thereby regulating memory formation. Both positive and negative regulators of translation play a critical role in learning and memory consolidation. The eukaryotic initiation factor 4E (eIF4E) homologous protein (4EHP, encoded by the gene Eif4e2) is a pivotal negative regulator of translation but its role in learning and memory is unknown. To address this gap in knowledge, we generated excitatory (glutamatergic: CaMKIIα-positive) and inhibitory (GABAergic: GAD65-positive) conditional knockout mice for 4EHP, which were analyzed in various behavioral memory tasks. Knockout of 4EHP in Camk2a-expressing neurons (4EHP-cKOexc) did not impact long-term memory in either contextual fear conditioning or Morris water maze tasks. Similarly, long-term contextual fear memory was not altered in Gad2-directed 4EHP knockout mice (4EHP-cKOinh). However, when subjected to a short-term T-maze working memory task, both mouse models exhibited impaired cognition. We therefore tested the hypothesis that de novo protein synthesis plays a direct role in working memory. We discovered that phosphorylation of ribosomal protein S6, a measure of mTORC1 activity, is dramatically reduced in the CA1 hippocampus of 4EHP-cKOexc mice. Consistently, genetic reduction of mTORC1 activity in either excitatory or inhibitory neurons was sufficient to impair working memory. Taken together, these findings indicate that translational control by 4EHP and mTORC1 in both excitatory and inhibitory neurons are necessary for working memory

Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration

Autophagosomes primarily mediate turnover of cytoplasmic proteins or organelles to provide nutrients and eliminate damaged proteins. In neurons, autophagosomes form in distal axons and are trafficked retrogradely to fuse with lysosomes in the soma. Although defective neuronal autophagy is associated with neurodegeneration, the function of neuronal autophagosomes remains incompletely understood. We show that in neurons, autophagosomes promote neuronal complexity and prevent neurodegeneration in vivo via retrograde transport of brain-derived neurotrophic factor (BDNF)-activated TrkB receptors. p150Glued/dynactin-dependent transport of TrkB-containing autophagosomes requires their association with the endocytic adaptor AP-2, an essential protein complex previously thought to function exclusively in clathrin-mediated endocytosis. These data highlight a novel non-canonical function of AP-2 in retrograde transport of BDNF/TrkB-containing autophagosomes in neurons and reveal a causative link between autophagy and BDNF/TrkB signalling

Phosphorylation of rpS6 at Ser235/236 in the piriform cortex and amygdala upon kainic acid-induced status epilepticus and rapamycin treatment.

<p>(<b><i>A</i></b>) Representative images of piriform cortex and amygdala sections immunohistochemically stained for P-S6 in control animals and in animals 2 and 24 h after KA injection. Dotted lines on the left panels outline piriform cortex and amygdala. Panels on the right represent higher-magnification of cortical and amygdalar regions boxed with the solid line on the major view (left panels). (<b><i>B</i></b>) Representative images of piriform cortex and amygdala sections immunohistochemically stained for P-S6 in rapamycin- and rapamycin+KA-treated animals 2 and 24 h after KA treatment. Dotted lines on the left panels outline piriform cortex and amygdala. Panels on the right represent higher-magnification of cortical and amygdalar regions boxed on the major view (left panels). RA – chronic rapamycin treatment. Arrows indicate P-S6-positive neuronal-like cells. Arrowheads show glial-like P-S6-immunoreactive cells. Scale bar = 200 µm (left panel). Scale bar = 20 µm (right panel).</p

Kainic acid-induced changes in the subcellular distribution of mTOR phosphorylation at Ser2448.

<p>(<b><i>A</i></b>) Immunohistochemical analysis of P-mTOR expression in the hippocampus in control rats and in rats 2 and 24 h after kainic acid (KA)-induced status epilepticus. Scale bar = 200 µm. (<b><i>B</i></b>) Images of single cells of the dentate gyrus (DG) hilus (<i>upper panel</i>) and cortex <i>(lower panel</i>) of the animals described in <i>A</i>. Scale bar = 10 µm. (<b><i>C</i></b>) Representative results of Western blot analysis of hippocampal P-mTOR and mTOR levels in the control animals and in animals 2 h after KA injection. (<b><i>D</i></b>) Representative confocal images of double fluorescence staining with antibodies against P-mTOR (green) and nuclear dye Hoechst 33258 (blue) of the CA1 and DG regions of the hippocampus in control rats and in rats 2 h after KA administration. Arrowheads indicate double-stained nuclei. Scale bar = 10 µm.</p

Chronic rapamycin treatment influences seizure susceptibility, KA-induced mortality and gross brain morphology.

<p>(<b><i>A, B, C</i></b>) Analysis of mean latency from kainic acid (KA) injection to stage 3 seizures (<i>B</i>) percentage of animals which developed seizures (<i>C</i>) and mortality, induced by KA in rats treated for either one week (short treatment) or four weeks (long treatment) with vehicle (short treatment: <i>n</i> = 20; long treatment: <i>n</i> = 14) or RA (short treatment: <i>n</i> = 20; long treatment: <i>n</i> = 36). Error bars represent the standard deviation. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; Mann Whitney U-test. (<b><i>D</i></b>) Representative images of Nissl-stained hippocampal sections in animals after long treatment with vehicle (<i>n</i> = 7) or RA (<i>n</i> = 8). Scale bar = 200 µm. (<b><i>E</i></b><i>)</i> Analysis of changes in the hippocampus, ventricular and entire hemisphere areas in rats treated as in <i>D</i>. Error bars represent the standard deviation. *<i>p</i><0.05, **<i>p</i><0.01; Mann Whitney U-test. (<b><i>F</i></b>) Representative images of Nissl-stained hippocampal sections in animals after short treatment with vehicle (<i>n</i> = 8) or RA (<i>n</i> = 6). Scale bar = 200 µm. (<b><i>G</i></b><i>)</i> Analysis of changes in the hippocampus, ventricular and entire hemisphere areas in rats treated as in <i>F</i>. Error bars represent the standard deviation.</p

Quantitative analysis of kainic acid-induced changes in mTOR phosphorylation at Ser2448.

<p>(<b><i>A</i></b>) Representative micrographs of Nissl-stained brain sections with an outline of brain regions used for P-mTOR immunoreactivity (IR) semi-Q-IHC analysis. Areas of cell body layers for mean optical density measurements are surrounded with yellow line while areas used for hippocampal and piriform cortex neuropil analysis are contoured with black line. (<b><i>B</i></b>) Quantification of cellular layer and neuropil P-mTOR immunoreactivity in hippocampi and piriform cortex of control animals (<i>n</i> = 9) and animals treated with kainic acid (KA) and evaluated at 2 h (<i>n</i> = 6) and 24 h (<i>n</i> = 6) post KA. Error bars represent the standard deviation. Asterisks indicate significant changes in neuropil P-mTOR IR vs. control, *<i>p</i><0.05, **<i>p</i><0.01; Mann Whitney U-test. (<b><i>C</i></b>) Quantification of ratio of cellular layer P-mTOR IR to neuropil P-mTOR IR. Error bars represent the standard deviation. *<i>p</i><0.05, **<i>p</i><0.01; Mann Whitney U-test.</p

Phosphorylation of rpS6 at Ser235/236 increases with kainic acid-induced status epilepticus and depends on mTOR activity: a quantitative Western blot analysis.

<p>Quantitative Western blot analysis of phosphorylated rpS6 (P-S6) levels in the hippocampus and cortices in control (Ctr) animals (<i>n</i> = 5), animals that received kainic acid (KA) treatment and were evaluated at 2 h (<i>n</i> = 5), animals that received rapamycin (RA) treatment for 4 weeks (<i>n</i> = 6), and animals that received both KA and rapamycin treatment (<i>n</i> = 7). The graphs represent relative P-S6 levels normalized to tubulin. Error bars represent the standard deviation. *<i>p</i><0.05, **<i>p</i><0.01; Mann-Whitney U test.</p