Specification of Neuronal Polarity Regulated by Local Translation of CRMP2 and Tau via the mTOR-p70S6K Pathway

Mammalian target of rapamycin (mTOR) is an important regulator of neuronal development and functions. Although it was reported recently that mTOR signaling is critical for neuronal polarity, the underlying mechanism remains unclear. Here, we describe the molecular pathway of mTOR-dependent axon specification, in which the collapsing response mediator protein 2 (CRMP2) and Tau are major downstream targets. The activity of mTOR effector 70-kDa ribosomal protein S6 kinase (p70S6K) specifically increases in the axon during neuronal polarity formation. The mTOR inhibitor rapamycin suppresses the translation of some neuronal polarity proteins, including CRMP2 and Tau, thereby inhibiting axon formation. In contrast, constitutively active p70S6K up-regulates the translation of these molecules, thus inducing multiple axons. Exogenous CRMP2 and Tau facilitate axon formation, even in the presence of rapamycin. In the 5′-untranslated region of Tau and CRMP2 mRNAs, we identified a 5′-terminal oligopyrimidine tract, which mediates mTOR-governed protein synthesis. The 5′-terminal oligopyrimidine tract sequences of CRMP2 and Tau mRNAs strongly contribute to the up-regulation of their translation in the axon in response to the axonal activation of the mTOR-p70S6K pathway. Taken together, we conclude that the local translation of CRMP2 and Tau, regulated by mTOR-p70S6K, is critical for the specification of neuronal polarity.To function, developing neurons must establish axonal-dendritic polarity. The polarization processes have been well studied in cultured hippocampal neurons (1, 2). They first form lamellipodia and small protrusion veils shortly after plating (stage 1) and then extend several immature neurites within 12–24 h (stage 2). One of the neurites rapidly elongates and becomes the axon, whereas the others become dendrites (stage 3). During these processes, a number of molecules coordinately control the axon specification (3). These molecules spatially regulate cytoskeletal networks of actin filaments and microtubules for specification and maintenance of a single axon.Mammalian target of rapamycin (mTOR)² is a serine/threonine protein kinase and mainly controls protein synthesis via phosphorylation of its downstream targets, such as eukaryotic translation initiation factor 4E (eIF-4E)-binding protein 1 (4E-BP1) and 70-kDa ribosomal S6 protein kinase (p70S6K) (4). In the nervous system, mTOR plays important roles in neuronal survival, dendritic arbor formation, synaptic plasticity, learning, and memory (5). Notably, in the regulation of synaptic plasticity, a number of mRNAs are locally translated via activation of the mTOR pathway in response to extracellular signals (5–7). Recently, two research groups reported that the mTOR pathway is also critical for the formation of neuronal polarity (8, 9). They proposed that the mTOR pathway controls the synthesis of polarity proteins, such as synapses of the amphid-detective (SAD) kinases (8) and Rap1 (9), and therefore regulates the neuronal polarity. However, the underlying molecular mechanism remains to be fully understood. Here, we demonstrate that p70S6K is necessary and sufficient for the mTOR-controlled axon formation as a downstream effector of mTOR. The mTOR-p70S6K pathway regulates translation of several neuronal polarity genes, including SAD kinases and Rap1. In particular, the expressions of collapsing response mediator protein 2 (CRMP2) and microtubule-associated protein Tau are highly dependent on mTOR-governed protein synthesis. We further identified a 5′-terminal oligopyrimidine (5′-TOP) tract, which mediates mTOR-governed protein synthesis (10), in the 5′-UTRs of CRMP2 and Tau mRNAs. In the axon of developing neurons, the translation of CRMP2 and Tau mRNAs is spatially controlled by the mTOR-p70S6K pathway via the 5′-TOP sequences, resulting in accumulation of CRMP2 and Tau proteins in the axon. Thus, our study substantially improves understanding of molecular mechanisms underlying the mTOR-dependent development of neuronal polarity.     

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca²⁺ concentration. Tn is composed of TnC, TnI, and TnT. Ca²⁺ binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca²⁺ activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca²⁺, which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca²⁺ NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca²⁺, there was little difference in the activation of ATPase activity between these preparations.Striated muscle thin filaments exist in equilibrium among multiple states. Ca²⁺ binding to the regulatory domain of troponin C (TnC)² along the thin filaments and strong cross-bridge interactions with thick filaments are thought to shift the equilibrium. Ca²⁺ binds to the regulatory domain of TnC, which regulates the interaction of troponin I (TnI) with actin-tropomyosin (Tm) and TnC (1–3). In the thin filaments, the inhibitory region of TnI (residues 104–115 of rabbit fast skeletal TnI (fsTnI) or 138–149 of mouse cardiac TnI (cTnI)) undergoes a structural transition depending on the Ca²⁺ state of TnC (4, 5). In the absence of Ca²⁺ at the regulatory site(s) of TnC, the inhibitory region interacts with actin to prevent activation of myosin ATPase activity. When Ca²⁺ binds to the regulatory site(s) of TnC, the switch region of TnI, which is located at the C terminus of the inhibitory region, interacts with the newly exposed hydrophobic patch of the N-terminal regulatory domain of TnC (6–8). This interaction causes the removal of the inhibitory region and the second actin-Tm binding region of TnI from the actin surface and allows actin to interact with myosin. In the presence of Ca²⁺ at the regulatory sites of TnC, the inhibitory region and the central helical region of TnC are mutually stabilized, according to the recent x-ray crystal structure of the core domain of the fsTn complex (9). The sequence variations in the N-terminal and the C-terminal regions of TnT, another component of the Tn complex, are known to alter the Ca²⁺ sensitivity of myofilament activity (10, 11). In addition, TnT is involved in the Ca²⁺-dependent interaction of the Tn complex with actin-Tm (12). However, the molecular mechanism whereby TnT participates in the Ca²⁺ regulation has not been established.There is evidence supporting the idea that each amino acid residue in the inhibitory region of TnI contributes differently and to a different degree to myofilament activities. One example is genetic mutations and phosphorylation of amino acid residues in the inhibitory region of cardiac TnI that cause the modification of myofilament activities. In hypertrophic or restrictive cardiomyopathy-linked mutations found in the inhibitory region, such as R142Q, L145Q, and R146G/Q/W mutations (mouse cTnI sequence number), induce Ca²⁺ sensitization of myofilament activities and an increase in ATPase/tension at low [Ca²⁺] (13, 14). Recently we reported that thin filaments reconstituted with R146G or R146W mutant cTnI bind Ca²⁺ tighter than those with cTnI(wt) (15). The Ca²⁺ sensitization may occur as a result of the destabilization of the off-state of the thin filaments due to the mutation introduced into the actin-Tm-interacting residue, i.e. Arg-146, of cTnI. On the other hand, Thr-144 is phosphorylated by protein kinase C (PKC) specifically, although the consequence of the PKC-dependent phosphorylation of Thr-144 has not yet been clearly defined. Pseudophosphorylation of Thr-144 was shown to cause Ca²⁺ desensitization in in vitro motility assays (16), whereas there is a report that indicates phosphorylation of Thr-144 sensitizes skinned cardiomyocytes to Ca²⁺ (17). Furthermore, Tachampa et al. reported that Thr-144 of cTnI is important for length-dependent activation of skinned cardiac muscle (18). Thus in each case presented above, a specific change in a single amino acid in the inhibitory region of TnI induced different and divergent effects on myofilament activities.Our aim of this study is to assess the functional contributions of the individual amino acid residues in the inhibitory region to the regulatory function. To assess the functional roles of the individual amino acid residues systematically, we used Ala scanning (19, 20). Ala substitution deletes all the interactions made by atoms beyond β-C yet does not alter the peptide backbone conformation, unless it is applied to Gly or Pro. Ala is one of the most abundant amino acids and is found in both buried and exposed positions. We found that almost the entire minimum inhibitory region of cTnI we investigated (Fig. 1) is important for both the inhibition and activation. Our data also indicate that the C-terminal part of the inhibitory region destabilizes the active state of the thin filaments. We also found that Thr-144 is involved in NEM-S1-dependent activation of ATPase activity in the absence of Ca²⁺.Open in a separate windowFIGURE 1.Inhibitory region of TnI. A, sequence comparison of the minimum inhibitory region from various vertebrates. The amino acid residues that are different from fsTnI are colored green in cardiac sequences. Note the amino acid sequence of the inhibitory region is highly conserved. Also the amino acid sequences of the minimum inhibitory region of the mutants we investigated in this study are shown. B, crystal structure of the inhibitory region and its surrounding region in chicken fsTn complex in the Ca²⁺-bound form (PDB: 1YTZ). TnC, pink; TnT, light blue; TnI, gray. The segment, corresponding to residues 143–149 of mouse cTnI, is colored red.     

Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

Differential gene expression in Giardia lamblia under oxidative stress: Significance in eukaryotic evolution

Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production.     

Metabolomic analysis of the effects of omeprazole and famotidine on aspirin-induced gastric injury

Gastric mucosal ulceration and gastric hemorrhage are frequently associated with treatment by non-steroid anti-inflammatory drugs (NSAIDs); however, no convenient biomarker-based diagnostic methods for these adverse reactions are currently available, requiring the use of endoscopic evaluation. We recently reported five biomarker candidates in serum which predict gastric injury induced by NSAIDs in rats, but were unable to clarify the mechanism of change in the levels of these biomarker candidates. In this study, we performed capillary electrophoresis–mass spectrometry-based metabolomic profiling in stomach and serum from rats in which gastric ulcer was induced by aspirin and prevented by co-administration of omeprazole and famotidine. Results showed drug-induced decreases in the levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, and O-acetyl carnitine in all animals administered aspirin. In contrast, aspirin-induced decreases in the level of 4-hydroxyproline were suppressed by co-administration of omeprazole and famotidine. We consider that these changes were due to the prevention of gastric ulcer and decrease in the amount of collagen in stomach tissue by omeprazole and famotidine, without prevention of the NSAID-induced depression of mitochondrial function. In addition, the decreases in 4-hydroxyproline in the stomach was also detectable as changes in the serum. While further study is needed to clarify limitations of indications and extrapolation to humans, this new serum biomarker candidate of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.