Phospho-Dependent Functional Modulation of GABAB Receptors by the Metabolic Sensor AMP-Dependent Protein Kinase

Kuramoto, Nobuyuki; Wilkins, Megan E.; Fairfax, Benjamin P.; Revilla-Sanchez, Raquel; Terunuma, Miho; Tamaki, Keisuke; Iemata, Mika; Warren, Noël A.; Couve, Andrés; Calver, Andrew R.; Horváth, Zsolt; Freeman, Katie B.; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward C.; Smart, Trevor G.; Pangalos, Menelas N.; Moss, Stephen J.

doi:10.1016/j.neuron.2006.12.015

Cited by 166 publications

(168 citation statements)

References 40 publications

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“…S1E). To confirm the in vivo ablation of the catalytic activity of AMPK, we used phospho-specific antibodies detecting two previously characterized AMPK protein substrates: serine 783 of the GABA B receptor 2 (19) and retinoblastoma (Rb) protein on serines 807 and 811 (10). Phosphorylation of both of these proteins is abolished in the P3 cortex of AMPKα-DcKO compared with control mice (Fig.…”

Section: Resultsmentioning

confidence: 99%

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Williams¹,

Courchet

Viollet

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

111

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Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the β1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic lossof-function approach producing AMPKα1/α2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner.A MP-activated kinase (AMPK) is a heterotrimeric serine/ threonine protein kinase composed of one catalytic subunit (encoded by α1 or α2 genes in mammals) and two regulatory subunits β and γ (encoded by β1 or β2 genes and γ1, γ2, or γ3 genes, respectively) (1-3). AMPK is an important metabolic sensor, activated by various forms of metabolic stress including low ATP: AMP ratios. AMPK has been implicated in a range of cell biological functions including cell polarity, autophagy, apoptosis, and cell migration (2-9). A recent study (10) suggested that the regulatory subunit, AMPKβ1, is critical for normal neurogenesis, neuronal differentiation, and neuronal survival during cortical development. However, to date there is no published evidence reporting the consequence of a genetic loss of function for the catalytic activity of mammalian AMPK in the mammalian nervous system. To assess the role of AMPKα during cortical development, we used transgenic mice that were ubiquitously inactivated for the AMPKα1 gene (AMPKα1 −/− ) (11) and conditionally inactivated for AMPKα2 (AMPKα2 F/F ) (12, 13). AMPKα2 was selectively deleted using the Emx1 Cre mouse line, which induces recombination only in dorsal telencephalic progenitors giving rise to all pyramidal projection neurons in the cortex, but not in ventral telencephalon-derived cortical GABAergic interneurons, which constitutes ∼25% of all cortical neurons (14). Surprisingly, we found no obvious defect of neurogenesis, neuronal migration, axon formation, or neuronal survival in AMPKα1/2-null cortex compared with control mice. On the basis of the profound di...

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Section: Resultsmentioning

confidence: 99%

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Williams¹,

Courchet

Viollet

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

111

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“…It has been reported that lithium inhibits PP2A activity [20,21], which has been shown to regulate phosphorylation of AMPK [31]. The observed effects of lithium on AMPK may also be related to the neuroprotective effects of lithium because AMPK activation is connected to cell survival in several systems [32][33][34]. Immunohistochemical data suggested that the subcellular localization of phospho-AMPK in "ethanol + lithium" group is different from that in control and lithium groups.…”

Section: Discussionmentioning

confidence: 99%

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

Chakraborty

Shimada

Mao

et al. 2008

Biochemical and Biophysical Research Communications

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Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanolinduced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and Nacylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

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“…Patch Clamp Electrophysiology-Membrane currents were recorded, using whole cell patch clamp, from single GIRK cells as described previously (24). Patch pipettes (3-5 M⍀) were filled with a solution containing (mM): 120 KCl, 2 MgCl 2 , 11 EGTA, 30 KOH, 10 HEPES, 1 CaCl 2 , 1 GTP, 2 ATP, 14 creatine phosphate, pH 7.0.…”

Section: Gaba B Receptor Containing the Btx-binding Site-com-mentioning

confidence: 99%

“…To construct GABA concentration-response relationships, the current (I) was measured in the presence of each concentration of GABA applied at 3-min intervals as described previously (24). The currents were normalized to the maximum GABA response (I max ) and the concentration response relationship fitted with the Hill equation (24).…”

Section: Gaba B Receptor Containing the Btx-binding Site-com-mentioning

confidence: 99%

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Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

Wilkins

Smart

2008

Journal of Biological Chemistry

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GABA B receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre-and postsynaptic sites, GABA B receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA B receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, ␣-bungarotoxin. By using the ␣-bungarotoxin binding site-tagged GABA B R1a subunit (R1a BBS ), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, ␣-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA B receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors. ␥-Aminobutyric acid (GABA)2 is the major inhibitory neurotransmitter in the central nervous system (CNS) activating ionotropic GABA A/C , as well as the metabotropic GABA B receptor. GABA B receptors are expressed in all major brain structures (1-3) and are important for synaptic plasticity as well as having therapeutic implications for epilepsy, pain, spasticity, drug addiction, schizophrenia, depression, and anxiety (4).The trafficking and cell surface mobility of ligand-gated GABA A receptors has been studied using reporter tags with electrophysiological (5) or imaging approaches (6, 7). However, the mobility and trafficking of extrasynaptic GABA B receptors has provided diverse results (8 -11). The GABA B receptor is a heterodimeric G-protein-coupled receptor (GPCR), requiring R1 and R2 subunits to co-assemble before trafficking to the cell surface to form functional receptors. The R1 subunit possesses an ER retention motif that is masked by binding to the R2 subunit (12-14). Although, generally, GPCRs are readily internalized from the cell surface following agonist activation and receptor phosphorylation (15-17); the GABA B receptor was thought to behave differently, being relatively stable in the cell membrane (8, 9). However, other reports indicate that agonist activation of GABA B receptors may promote internalization and/or rapid recycling (10,11,18). To address the topic of GABA B receptor trafficking, prior studies have used various techniques to monitor receptor movement, including: receptor biotinylation (8, 9); antibody labeling of extracellular GABA B receptor epitopes on live cells (9); as well as fluorescence recovery after photobleaching (FRAP) (10). These methods have therefore relied on the use of relatively large reporter molecules, such as antibodies. Although such studies have...

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Phospho-Dependent Functional Modulation of GABAB Receptors by the Metabolic Sensor AMP-Dependent Protein Kinase

Cited by 166 publications

References 40 publications

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

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