Graham H. Diering

Structure-Activity Analysis of Niclosamide Reveals Potential Role for Cytoplasmic pH in Control of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling

Background: mTORC1 is dysregulated in human disease, and there is an interest in the development of mTORC1 inhibitors. Niclosamide inhibits mTORC1 signaling, but its mode of action remains unclear. Results: Niclosamide extrudes protons from lysosomes, thus lowering cytoplasmic pH and inhibiting mTORC1 signaling. Conclusion: Cytoplasmic acidification inhibits mTORC1 signaling. Significance: Our findings may aid the design of niclosamide-based anticancer therapeutic agents. Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.

Homer1a drives homeostatic scaling-down of excitatory synapses during sleep

Synapse remodeling during sleep General activity and information processing while an animal is awake drive synapse strengthening. This is counterbalanced by weakening of synapses during sleep (see the Perspective by Acsády). De Vivo et al. used serial scanning electron microscopy to reconstruct axon-spine interface and spine head volume in the mouse brain. They observed a substantial decrease in interface size after sleep. The largest relative changes occurred among weak synapses, whereas strong ones remained stable. Diering et al. found that synapses undergo changes in synaptic glutamate receptors during the sleep-wake cycle, driven by the immediate early gene Homer1a. In awake animals, Homer1a accumulates in neurons but is excluded from synapses by high levels of noradrenaline. At the onset of sleep, noradrenaline levels decline, allowing Homer1a to move to excitatory synapses and drive synapse weakening. Science, this issue p. 457, p. 507; see also p. 511 Excitatory synapses are globally weakened during sleep. Sleep is an essential process that supports learning and memory by acting on synapses through poorly understood molecular mechanisms. Using biochemistry, proteomics, and imaging in mice, we find that during sleep, synapses undergo widespread alterations in composition and signaling, including weakening of synapses through removal and dephosphorylation of synaptic AMPA-type glutamate receptors. These changes are driven by the immediate early gene Homer1a and signaling from group I metabotropic glutamate receptors mGluR1/5. Homer1a serves as a molecular integrator of arousal and sleep need via the wake- and sleep-promoting neuromodulators, noradrenaline and adenosine, respectively. Our data suggest that homeostatic scaling-down, a global form of synaptic plasticity, is active during sleep to remodel synapses and participates in the consolidation of contextual memory.

Regulation of mTORC1 Signaling by pH

Background Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. Methodology/Findings We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1–TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1–TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. Conclusions This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.

Sorting Nexin 27 regulates basal and activity-dependent trafficking of AMPARs

Significance AMPA-type glutamate receptors (AMPARs) are principal regulators of synaptic signaling in the brain. Modulation of AMPA receptor activity, whether through changes in surface expression or conductance, contributes significantly to the dynamic nature of neuronal networks. AMPA receptor mediated-synaptic plasticity is thought to underlie learning and memory, as aberrant AMPAR trafficking contributes to impaired plasticity and to memory deficits. We demonstrate that the membrane trafficking and sorting protein, SNX27, regulates basal and activity dependent targeting of AMPA receptors to the neuronal surface. Elucidating the mechanism of receptor sorting mediated by SNX27 will not only further our understanding AMPAR-mediated changes in synaptic plasticity, but may ultimately shed light on the molecular mechanisms that govern learning. Activity-dependent changes in synaptic strength have long been postulated as cellular correlates of learning and memory. Long-term potentiation (LTP), a well characterized form of synaptic plasticity, is often expressed as an increase in the number of postsynaptic AMPA-type glutamate receptors (AMPARs). Although the precise molecular mechanisms governing LTP remain elusive, this study identifies one member of the sorting nexin family, Sorting Nexin 27 (SNX27), as a critical component in this process. The ability of sorting nexins to bind specific phospholipids as well as their propensity to form protein–protein complexes, points to a role for these proteins in membrane trafficking and protein sorting. Here, we demonstrate that SNX27 binds to AMPARs, and that this interaction is regulated in an activity-dependent manner. Furthermore, we provide evidence that SNX27 is synaptically enriched and its level of expression regulates targeting of AMPARs to the neuronal surface. Loss of SNX27 abolishes recruitment of surface AMPARs during chemical LTP. Collectively, our data suggest a role for SNX27 in modulating synaptic plasticity through regulated interaction with AMPARs.

Regulation of dendritic spine growth through activity-dependent recruitment of the brain-enriched Na+/H+ exchanger NHE5

pH homeostasis in neurons plays crucial roles in normal synaptic functions. It is found that the Na+/H+ exchanger NHE5 is targeted to the synapse on neuronal activation, regulates the synaptic pH, and controls the morphology of dendritic spines.

Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

The role of endosomal pH in neurite formation, one of the principal processes of neuronal differentiation, is unknown. This study shows that the neuron-enriched Na+/H+ exchanger NHE5 potently acidifies recycling endosomes and regulates TrkA trafficking, NGF-TrkA signaling, and neurite outgrowth.

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na+/H+ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.

Extensive phosphorylation of AMPA receptors in neurons

Significance Decades of research from many laboratories has established a model in which phosphorylation of the GluA1 AMPA-type glutamate receptor subunit plays a significant role modulating long-term potentiation and depression, homeostatic and neuromodulator-regulated plasticity, spatial memory, fear/extinction, and appetitive incentive learning. However, a recent study suggests that GluA1 phosphorylation is exceedingly low, even in synaptic fractions. Here, we address this controversy using in vitro and in vivo techniques. We find a large fraction of excitatory synapses are positive for phosphorylated GluA1. Moreover, phosphorylated species make up a significant fraction of the population and are highly responsive to numerous physiologically relevant stimuli. This characterization reaffirms a large body of work defining a prominent role of AMPA receptor phosphorylation in synapse biology and synaptic plasticity. Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein–protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1–containing AMPARs. Our results support the large body of research indicating a prominent role of GluA1 phosphorylation in synaptic plasticity.

Endosomal pH in neuronal signaling and synaptic transmission: role of Na + /H + exchanger NHE5

Neuronal precursor cells extend multiple neurites during development, one of which extends to form an axon whereas others develop into dendrites. Chemical stimulation of N-methyl D-aspartate (NMDA) receptor in fully-differentiated neurons induces projection of dendritic spines, small spikes protruding from dendrites, thereby establishing another layer of polarity within the dendrite. Neuron-enriched Na+/H+ exchanger NHE5 contributes to both neurite growth and dendritic spine formation. In resting neurons and neuro-endocrine cells, neuron-enriched NHE5 is predominantly associated with recycling endosomes where it colocalizes with nerve growth factor (NGF) receptor TrkA. NHE5 potently acidifies the lumen of TrkA-positive recycling endosomes and regulates cell-surface targeting of TrkA, whereas chemical stimulation of NMDA receptors rapidly recruits NHE5 to dendritic spines, alkalinizes dendrites and down-regulates the dendritic spine formation. Possible roles of NHE5 in neuronal signaling via proton movement in subcellular compartments are discussed.

Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover

Significance The majority of cellular proteins undergo rapid degradation and synthesis to minimize the toxic effect to cells and tissues and to guarantee normal cellular functions. It has been appreciated that proteins with longer half-lives exist in certain cells and tissues. Here we identify synaptic long-lived proteins by high-resolution mass spectrometry. In general, synaptic proteins exhibit slower turnover than cytosolic proteins, and synaptic protein turnover from mouse brain is enhanced by enriched environment exposure. Moreover, protein half-lives are dynamically regulated during changes in neuronal activity. These findings demonstrate the existence of long-lived proteins in synapses in the brain and support a potential role for them in synaptic plasticity and learning and memory. Memory formation is believed to result from changes in synapse strength and structure. While memories may persist for the lifetime of an organism, the proteins and lipids that make up synapses undergo constant turnover with lifetimes from minutes to days. The molecular basis for memory maintenance may rely on a subset of long-lived proteins (LLPs). While it is known that LLPs exist, whether such proteins are present at synapses is unknown. We performed an unbiased screen using metabolic pulse-chase labeling in vivo in mice and in vitro in cultured neurons combined with quantitative proteomics. We identified synaptic LLPs with half-lives of several months or longer. Proteins in synaptic fractions generally exhibited longer lifetimes than proteins in cytosolic fractions. Protein turnover was sensitive to pharmacological manipulations of activity in neuronal cultures or in mice exposed to an enriched environment. We show that synapses contain LLPs that may underlie stabile long-lasting changes in synaptic structure and function.