RESUMEN
Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Potenciación a Largo Plazo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/fisiología , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Optogenética , Fosforilación , Unión ProteicaRESUMEN
Learning and memory require coordinated structural and functional plasticity at neuronal glutamatergic synapses located on dendritic spines. Here, we investigated how the endoplasmic reticulum (ER) controls postsynaptic Ca2+ signaling and long-term potentiation of dendritic spine size, i.e., sLTP that accompanies functional strengthening of glutamatergic synaptic transmission. In most ER-containing (ER+) spines, high-frequency optical glutamate uncaging (HFGU) induced long-lasting sLTP that was accompanied by a persistent increase in spine ER content downstream of a signaling cascade engaged by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter being activated by stromal interaction molecule 1 (STIM1) in response to ER Ca2+ release. In contrast, HFGU stimulation of ER-lacking (ER-) spines expressed only transient sLTP and exhibited weaker Ca2+ signals noticeably lacking Orai1 and ER contributions. Consistent with spine ER regulating structural metaplasticity, delivery of a second stimulus to ER- spines induced ER recruitment along with persistent sLTP, whereas ER+ spines showed no additional increases in size or ER content in response to sequential stimulation. Surprisingly, the physical interaction between STIM1 and Orai1 induced by ER Ca2+ release, but not the resulting Ca2+ entry through Orai1 channels, proved necessary for the persistent increases in both spine size and ER content required for expression of long-lasting late sLTP.
Asunto(s)
Canales de Calcio Tipo L , Espinas Dendríticas , Retículo Endoplásmico , Plasticidad Neuronal , Proteína ORAI1 , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 1/genética , Retículo Endoplásmico/metabolismo , Espinas Dendríticas/metabolismo , Animales , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Plasticidad Neuronal/fisiología , Canales de Calcio Tipo L/metabolismo , Potenciación a Largo Plazo/fisiología , Señalización del Calcio/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Calcio/metabolismo , Ratones , Transducción de Señal/fisiología , RatasRESUMEN
Neuronal excitatory synapses are primarily located on small dendritic protrusions called spines. During synaptic plasticity underlying learning and memory, Ca2+ influx through postsynaptic NMDA-type glutamate receptors (NMDARs) initiates signaling pathways that coordinate changes in dendritic spine structure and synaptic function. During long-term potentiation (LTP), high levels of NMDAR Ca2+ influx promote increases in both synaptic strength and dendritic spine size through activation of Ca2+-dependent protein kinases. In contrast, during long-term depression (LTD), low levels of NMDAR Ca2+ influx promote decreased synaptic strength and spine shrinkage and elimination through activation of the Ca2+-dependent protein phosphatase calcineurin (CaN), which is anchored at synapses via the scaffold protein A-kinase anchoring protein (AKAP)150. In Alzheimer's disease (AD) the pathological agent amyloid-ß (Aß) may impair learning and memory through biasing NMDAR Ca2+ signaling pathways toward LTD and spine elimination. By employing AKAP150 knock-in mice of both sexes with a mutation that disrupts CaN anchoring to AKAP150, we revealed that local, postsynaptic AKAP-CaN-LTD signaling was required for Aß-mediated impairment of NMDAR synaptic Ca2+ influx, inhibition of LTP, and dendritic spine loss. Additionally, we found that Aß acutely engages AKAP-CaN signaling through activation of G protein-coupled metabotropic glutamate receptor 1 (mGluR1) leading to dephosphorylation of NMDAR-GluN2B subunits, which decreases Ca2+ influx to favor LTD over LTP, and cofilin, which promotes F-actin severing to destabilize dendritic spines. These findings reveal a novel interplay between NMDAR and mGluR1 signaling that converges on AKAP-anchored CaN to coordinate dephosphorylation of postsynaptic substrates linked to multiple aspects of Aß-mediated synaptic dysfunction.Significance Statement Understanding mechanisms of synaptic dysfunction in Alzheimer's disease (AD) is pivotal for therapeutic advances. Amyloid-ß oligomers (Aßo), primary culprits in AD pathology, disrupt critical synaptic plasticity mechanisms, leading to enhanced LTD and synaptic loss. However, the underlying signaling pathways remain elusive. Calcineurin (CaN), localized by AKAP79/150 at synapses, plays a key role in LTD formation. Inhibition of CaN mitigates Aßo-induced synaptic deficits, implicating its involvement in AD pathology. Our study shows that AKAP-anchored CaN is critical in acute Aßo-mediated inhibition of NMDAR Ca2+ signaling and dendritic spine loss. Additionally, we identify mGluR1 as an upstream regulator of these Aßo-induced deficits, highlighting several potential therapeutic targets for AD-related synaptic pathology.
RESUMEN
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Asunto(s)
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Animales , Sistema Nervioso Central , Ácido Glutámico , Humanos , Neurotransmisores , Receptores Ionotrópicos de Glutamato/genéticaRESUMEN
The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic ß-cell, which secretes insulin upon glucose stimulation. Leptin is known to suppress glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the ß-cell surface, which increases K+ conductance and causes ß-cell hyperpolarization. We have previously shown that leptin-induced KATP channel trafficking requires protein kinase A (PKA)-dependent actin remodeling. However, whether PKA is a downstream effector of leptin signaling or PKA plays a permissive role is unknown. Using FRET-based reporters of PKA activity, we show that leptin increases PKA activity at the cell membrane and that this effect is dependent on N-methyl-D-aspartate receptors, CaMKKß, and AMPK, which are known to be involved in the leptin signaling pathway. Genetic knockdown and rescue experiments reveal that the increased PKA activity upon leptin stimulation requires the membrane-targeted PKA-anchoring protein AKAP79/150, indicating that PKA activated by leptin is anchored to AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, leads to increased surface KATP channels even in the absence of leptin stimulation. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate KATP channel trafficking in ß-cells, hence insulin secretion. The study further advances our knowledge of the downstream signaling events that may be targeted to restore insulin secretion regulation in ß-cells defective in leptin signaling, such as those from obese individuals with type 2 diabetes.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Secretoras de Insulina/metabolismo , Canales KATP/metabolismo , Leptina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Calcineurina/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Línea Celular , Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Glucosa/metabolismo , Homeostasis , Humanos , Insulina/metabolismo , Secreción de Insulina , Leptina/metabolismo , Fosforilación , Cultivo Primario de Células , Transporte de Proteínas , Transducción de SeñalRESUMEN
Patterns of postsynaptic activity that induce long-term potentiation of fast excitatory transmission at glutamatergic synapses between hippocampal neurons cause enlargement of the dendritic spine and promote growth in spine endoplasmic reticulum (ER) content. Such postsynaptic activity patterns also impact Ca2+ signaling in the adjoining dendritic shaft, in a zone centered on the spine-shaft junction and extending â¼10-20 µm in either direction along the shaft. Comparing this specialized zone in the shaft with the dendrite in general, plasticity-inducing stimulation of a single spine causes more profound depletion of Ca2+ stores in the ER, a greater degree of interaction between stromal interaction molecule 1 (STIM1) and L-type Ca2+ channels, and thus stronger STIM1 inhibition of these channels. Here we show that the length of this zone along the dendritic axis can be approximately doubled through the neuromodulatory action of ß-adrenergic receptors (ßARs). The mechanism of ßAR enlargement of the zone arises from protein kinase A-mediated enhancement of L-type Ca2+ current, which in turn lowers [Ca2+]ER through ryanodine receptor-dependent Ca2+-induced Ca2+ release and activates STIM1 feedback inhibition of L-type Ca2+ channels. An important function of this dendritic zone is to support crosstalk between spines along its length such that spines neighboring a strongly stimulated spine are enabled to undergo structural plasticity in response to stimulation that would otherwise be subthreshold for spine structural plasticity. This form of crosstalk requires L-type Ca2+ channel current to activate STIM1, and ßAR activity extends the range along the shaft over which such spine-to-spine communication can occur.
Asunto(s)
Calcio/metabolismo , Dendritas/fisiología , Espinas Dendríticas/fisiología , Receptor Cross-Talk/fisiología , Transducción de Señal , Sinapsis/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Retículo Endoplásmico/metabolismo , Femenino , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/fisiología , Molécula de Interacción Estromal 1/metabolismo , Potenciales Sinápticos/fisiologíaRESUMEN
Neuronal information processing requires multiple forms of synaptic plasticity mediated by NMDARs and AMPA-type glutamate receptors (AMPARs). These plasticity mechanisms include long-term potentiation (LTP) and long-term depression (LTD), which are Hebbian, homosynaptic mechanisms locally regulating synaptic strength of specific inputs, and homeostatic synaptic scaling, which is a heterosynaptic mechanism globally regulating synaptic strength across all inputs. In many cases, LTP and homeostatic scaling regulate AMPAR subunit composition to increase synaptic strength via incorporation of Ca2+-permeable receptors (CP-AMPAR) containing GluA1, but lacking GluA2, subunits. Previous work by our group and others demonstrated that anchoring of the kinase PKA and the phosphatase calcineurin (CaN) to A-kinase anchoring protein (AKAP) 150 play opposing roles in regulation of GluA1 Ser845 phosphorylation and CP-AMPAR synaptic incorporation during hippocampal LTP and LTD. Here, using both male and female knock-in mice that are deficient in PKA or CaN anchoring, we show that AKAP150-anchored PKA and CaN also play novel roles in controlling CP-AMPAR synaptic incorporation during homeostatic plasticity in hippocampal neurons. We found that genetic disruption of AKAP-PKA anchoring prevented increases in Ser845 phosphorylation and CP-AMPAR synaptic recruitment during rapid homeostatic synaptic scaling-up induced by combined blockade of action potential firing and NMDAR activity. In contrast, genetic disruption of AKAP-CaN anchoring resulted in basal increases in Ser845 phosphorylation and CP-AMPAR synaptic activity that blocked subsequent scaling-up by preventing additional CP-AMPAR recruitment. Thus, the balanced, opposing phospho-regulation provided by AKAP-anchored PKA and CaN is essential for control of both Hebbian and homeostatic plasticity mechanisms that require CP-AMPARs.SIGNIFICANCE STATEMENT Neuronal circuit function is shaped by multiple forms of activity-dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts strength of individual synapses and homeostatic plasticity that adjusts overall strength of all synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR regulation by the kinase PKA and phosphatase CaN coanchored to the scaffold protein AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic strength during homeostatic plasticity. These novel findings significantly expand our understanding of homeostatic plasticity mechanisms and further emphasize how intertwined they are with LTP and LTD.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/fisiología , Homeostasis/genética , Homeostasis/fisiología , Plasticidad Neuronal/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiología , Receptores AMPA/genética , Receptores AMPA/fisiología , Sinapsis/fisiología , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Fenómenos Electrofisiológicos/fisiología , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Técnicas de Sustitución del Gen , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Plasticidad Neuronal/fisiología , Cultivo Primario de Células , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiología , Reclutamiento Neurofisiológico/genética , Reclutamiento Neurofisiológico/fisiologíaRESUMEN
Calcineurin (CaN) phosphatase signaling is regulated by targeting CaN to substrates, inhibitors, and scaffold proteins containing docking motifs with the consensus sequence of PxIxIT. Here, we identify the docking of CaN to the γ isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Because of alternative splicing of a single exon within the N-terminal domain, MKK7γ encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7α or ß We found that MKK7γ bound directly to CaN through this PIIVIT motif in vitro, immunoprecipitated with CaN from cell extracts, and exhibited fluorescence resonance energy transfer (FRET) with CaN in the cytoplasm but not in the nucleus of living cells. In contrast, MKK7α and ß exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, the inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7γ but not MKK7ß Deletion of the MKK7γ PIIVIT motif eliminated FRET with CaN and promoted MKK7γ redistribution to the nucleus; however, the inhibition of CaN activity did not alter MKK7γ localization, indicating that MKK7γ cytoplasmic retention by CaN is phosphatase activity independent. Finally, the inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7γ mRNA, enhances JNK activation. Overall, we conclude that the MKK7γ-specific PxIxIT motif promotes high-affinity CaN binding that could promote novel cross talk between CaN and JNK signaling by limiting MKK7γ phosphorylation and restricting its localization to the cytoplasm.
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MAP Quinasa Quinasa 7/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica/fisiología , Isoformas de Proteínas/metabolismo , Empalme Alternativo/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
Both long-term potentiation (LTP) and depression (LTD) of excitatory synapse strength require the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and its autonomous activity generated by Thr-286 autophosphorylation. Additionally, LTP and LTD are correlated with dendritic spine enlargement and shrinkage that are accompanied by the synaptic accumulation or removal, respectively, of the AMPA-receptor regulatory scaffold protein A-kinase anchoring protein (AKAP) 79/150. We show here that the spine shrinkage associated with LTD indeed requires synaptic AKAP79/150 removal, which in turn requires CaMKII activity. In contrast to normal CaMKII substrates, the substrate sites within the AKAP79/150 N-terminal polybasic membrane-cytoskeletal targeting domain were phosphorylated more efficiently by autonomous compared with Ca2+/CaM-stimulated CaMKII activity. This unusual regulation was mediated by Ca2+/CaM binding to the substrate sites resulting in protection from phosphorylation in the presence of Ca2+/CaM, a mechanism that favors phosphorylation by prolonged, weak LTD stimuli versus brief, strong LTP stimuli. Phosphorylation by CaMKII inhibited AKAP79/150 association with F-actin; it also facilitated AKAP79/150 removal from spines but was not required for it. By contrast, LTD-induced spine removal of AKAP79/150 required its depalmitoylation on two Cys residues within the N-terminal targeting domain. Notably, such LTD-induced depalmitoylation was also blocked by CaMKII inhibition. These results provide a mechanism how CaMKII can indeed mediate not only LTP but also LTD through regulated substrate selection; however, in the case of AKAP79/150, indirect CaMKII effects on palmitoylation are more important than the effects of direct phosphorylation. Additionally, our results provide the first direct evidence for a function of the well-described AKAP79/150 trafficking in regulating LTD-induced spine shrinkage.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Lipoilación , Potenciación a Largo Plazo , Depresión Sináptica a Largo Plazo , Procesamiento Proteico-Postraduccional , Columna Vertebral/metabolismo , Sinapsis/metabolismo , Animales , Humanos , Columna Vertebral/patología , Sinapsis/patologíaRESUMEN
Phosphorylation and dephosphorylation of AMPA-type ionotropic glutamate receptors (AMPARs) by kinases and phosphatases and interactions with scaffold proteins play essential roles in regulating channel biophysical properties and trafficking events that control synaptic strength during NMDA receptor-dependent synaptic plasticity, such as LTP and LTD. We previously demonstrated that palmitoylation of the AMPAR-linked scaffold protein A-kinase anchoring protein (AKAP) 79/150 is required for its targeting to recycling endosomes in dendrites, where it regulates exocytosis from these compartments that is required for LTP-stimulated enlargement of postsynaptic dendritic spines, delivery of AMPARs to the plasma membrane, and maintenance of synaptic potentiation. Here, we report that the recycling endosome-resident palmitoyl acyltransferase DHHC2 interacts with and palmitoylates AKAP79/150 to regulate these plasticity signaling mechanisms. In particular, RNAi-mediated knockdown of DHHC2 expression in rat hippocampal neurons disrupted stimulation of exocytosis from recycling endosomes, enlargement of dendritic spines, AKAP recruitment to spines, and potentiation of AMPAR-mediated synaptic currents that occur during LTP. Importantly, expression of a palmitoylation-independent lipidated AKAP mutant in DHHC2-deficient neurons largely restored normal plasticity regulation. Thus, we conclude that DHHC2-AKAP79/150 signaling is an essential regulator of dendritic recycling endosome exocytosis that controls both structural and functional plasticity at excitatory synapses.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Aciltransferasas/metabolismo , Endosomas/metabolismo , Exocitosis , Potenciación a Largo Plazo , Aciltransferasas/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Espinas Dendríticas/metabolismo , Femenino , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Lipoilación , Masculino , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismo , Sinapsis/fisiologíaRESUMEN
Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aß) 1-42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, includingN-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aß(1-42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aß-induced synaptic dysfunction, but we recently found that Aß(1-42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aß(1-42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aß, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aß(1-42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aß(1-42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5-1.0µM Aß(1-42) or 1-5µM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aßon LTP and synapse loss, suggesting that Aßinduces acute and chronic synaptic dysfunction in part through inhibiting Eg5.
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Péptidos beta-Amiloides/toxicidad , Amiloide/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Hipocampo/efectos de los fármacos , Cinesinas/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Amiloide/metabolismo , Animales , Células Cultivadas , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/patología , Técnicas In Vitro , Cinesinas/metabolismo , Cinética , Potenciación a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Pirimidinas/toxicidad , Tionas/toxicidadRESUMEN
A central theme in nervous system function is equilibrium: synaptic strengths wax and wane, neuronal firing rates adjust up and down, and neural circuits balance excitation with inhibition. This push/pull regulatory theme carries through to the molecular level at excitatory synapses, where protein function is controlled through phosphorylation and dephosphorylation by kinases and phosphatases. However, these opposing enzymatic activities are only part of the equation as scaffolding interactions and assembly of multi-protein complexes are further required for efficient, localized synaptic signaling. This review will focus on coordination of postsynaptic serine/threonine kinase and phosphatase signaling by scaffold proteins during synaptic plasticity.
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Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transmisión Sináptica/fisiología , Animales , Humanos , Fosforilación/fisiologíaRESUMEN
Enhanced arterial tone is a leading cause of vascular complications during diabetes. Voltage-gated K(+) (KV) channels are key regulators of vascular smooth muscle cells (VSMCs) contractility and arterial tone. Whether impaired KV channel function contributes to enhance arterial tone during diabetes is unclear. Here, we demonstrate a reduction in KV-mediated currents (IKv) in VSMCs from a high fat diet (HFD) mouse model of type 2 diabetes. In particular, IKv sensitive to stromatoxin (ScTx), a potent KV2 blocker, were selectively reduced in diabetic VSMCs. This was associated with decreased KV2-mediated regulation of arterial tone and suppression of the KV2.1 subunit mRNA and protein in VSMCs/arteries isolated from HFD mice. We identified protein kinase A anchoring protein 150 (AKAP150), via targeting of the phosphatase calcineurin (CaN), and the transcription factor nuclear factor of activated T-cells c3 (NFATc3) as required determinants of KV2.1 suppression during diabetes. Interestingly, substantial reduction in transcript levels for KV2.1 preceded down-regulation of large conductance Ca(2+)-activated K(+) (BKCa) channel ß1 subunits, which are ultimately suppressed in chronic hyperglycemia to a similar extent. Together, our study supports the concept that transcriptional suppression of KV2.1 by activation of the AKAP150-CaN/NFATc3 signaling axis contributes to enhanced arterial tone during diabetes.
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Arterias/fisiología , Diabetes Mellitus Experimental/metabolismo , Regulación hacia Abajo , Tono Muscular/fisiología , Canales de Potasio Shab/fisiología , Animales , Diabetes Mellitus Experimental/fisiopatología , Ratones , Ratones Endogámicos C57BLRESUMEN
Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteína Quinasa C/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Encéfalo/metabolismo , Citoplasma/metabolismo , Silenciador del Gen , Glutatión Transferasa/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Ligandos , Sustancias Macromoleculares , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Isoformas de Proteínas , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular , Transducción de SeñalRESUMEN
Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.
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Proteínas de Anclaje a la Quinasa A/fisiología , Glucosa/metabolismo , Homeostasis/fisiología , Resistencia a la Insulina/genética , Proteínas de la Membrana/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Secuencias de Aminoácidos , Animales , Calcineurina/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , AMP Cíclico/fisiología , Glucosa/farmacología , Homeostasis/efectos de los fármacos , Insulina/metabolismo , Insulina/farmacología , Secreción de Insulina , Insulinoma/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Hígado/enzimología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Músculo Esquelético/enzimología , Neoplasias Pancreáticas/patología , Mapeo de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Eliminación de Secuencia , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
RATIONALE: Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes mellitus may arise from impaired large-conductance Ca(2+)-activated K(+) (BKCa) channel function. The scaffolding protein A-kinase anchoring protein 150 (AKAP150) is a key regulator of calcineurin (CaN), a phosphatase known to modulate the expression of the regulatory BKCa ß1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes mellitus is unknown. OBJECTIVE: To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa ß1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes mellitus. METHODS AND RESULTS: We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/nuclear factor of activated T-cells c3 (NFATc3) activation, and resistance artery constriction in hyperglycemic animals on high-fat diet. Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. d-glucose-dependent suppression of BKCa channel ß1 subunits required Ca(2+) influx via voltage-gated L-type Ca(2+) channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, high-fat diet mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa ß1 subunits and attenuated high-fat diet-induced elevation in arterial blood pressure. CONCLUSIONS: Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes mellitus.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Diabetes Mellitus Experimental/metabolismo , Hiperglucemia/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Vasoconstricción/fisiología , Proteínas de Anclaje a la Quinasa A/genética , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Grasas de la Dieta/farmacología , Técnicas de Sustitución del Gen , Hiperglucemia/genética , Hiperglucemia/fisiopatología , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Factores de Transcripción NFATC/metabolismo , Péptidos/farmacología , Transducción de Señal/fisiología , Toxinas Biológicas/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
Alzheimer's Disease (AD) is associated with brain accumulation of synaptotoxic amyloid-ß (Aß) peptides produced by the proteolytic processing of amyloid precursor protein (APP). Cognitive impairments associated with AD correlate with dendritic spine and excitatory synapse loss, particularly within the hippocampus. In rodents, soluble Aß oligomers impair hippocampus-dependent learning and memory, promote dendritic spine loss, inhibit NMDA-type glutamate receptor (NMDAR)-dependent long-term potentiation (LTP), and promote synaptic depression (LTD), at least in part through activation of the Ca2+-CaM-dependent phosphatase calcineurin (CaN). Yet, questions remain regarding Aß-dependent postsynaptic CaN signaling specifically at the synapse to mediate its synaptotoxicity. Here, we use pharmacologic and genetic approaches to demonstrate a role for postsynaptic signaling via A kinase-anchoring protein 150 (AKAP150)-scaffolded CaN in mediating Aß-induced dendritic spine loss in hippocampal neurons from rats and mice of both sexes. In particular, we found that Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs), which were previously shown to signal through AKAP-anchored CaN to promote both LTD and Aß-dependent inhibition of LTP, are also required upstream of AKAP-CaN signaling to mediate spine loss via overexpression of APP containing multiple mutations linked to familial, early-onset AD and increased Aß production. In addition, we found that the CaN-dependent nuclear factor of activated T-cells (NFAT) transcription factors are required downstream to promote Aß-mediated dendritic spine loss. Finally, we identified the E3-ubiquitin ligase Mdm2, which was previously linked to LTD and developmental synapse elimination, as a downstream NFAT target gene upregulated by Aß whose enzymatic activity is required for Aß-mediated spine loss.Significance Statement Impaired hippocampal function and synapse loss are hallmarks of AD linked to Aß oligomers. Aß exposure acutely blocks hippocampal LTP and enhances LTD and chronically leads to dendritic spine synapse loss. In particular, Aß hijacks normal plasticity mechanisms, biasing them toward synapse weakening/elimination, with previous studies broadly linking CaN phosphatase signaling to this synaptic dysfunction. However, we do not understand how Aß engages signaling specifically at synapses. Here we elucidate a synapse-to-nucleus signaling pathway coordinated by the postsynaptic scaffold protein AKAP150 that is activated by Ca2+ influx through CP-AMPARs and transduced to nucleus by CaN-NFAT signaling to transcriptionally upregulate the E3-ubiquitin ligase Mdm2 that is required for Aß-mediated spine loss. These findings identify Mdm2 as potential therapeutic target for AD.
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Palmitoylation and depalmitoylation represent dichotomic processes by which a labile posttranslational lipid modification regulates protein trafficking and degradation. The depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), is associated with the devastating pediatric neurodegenerative condition, infantile neuronal ceroid lipofuscinosis (CLN1). CLN1 is characterized by the accumulation of autofluorescent lysosomal storage material (AFSM) in neurons and robust neuroinflammation. Converging lines of evidence suggest that in addition to cellular waste accumulation, the symptomology of CLN1 corresponds with disruption of synaptic processes. Indeed, loss of Ppt1 function in cortical neurons dysregulates the synaptic incorporation of the GluA1 AMPA receptor (AMPAR) subunit during a type of synaptic plasticity called synaptic scaling. However, the mechanisms causing this aberration are unknown. Here, we used the Ppt1-/- mouse model (both sexes) to further investigate how Ppt1 regulates synaptic plasticity and how its disruption affects downstream signaling pathways. To this end, we performed a palmitoyl-proteomic screen, which provoked the discovery that Akap5 is excessively palmitoylated at Ppt1-/- synapses. Extending our previous data, in vivo induction of synaptic scaling, which is regulated by Akap5, caused an excessive upregulation of GluA1 in Ppt1-/- mice. This synaptic change was associated with exacerbated disease pathology. Furthermore, the Akap5- and inflammation-associated transcriptional regulator, nuclear factor of activated T cells (NFAT), was sensitized in Ppt1-/- cortical neurons. Suppressing the upstream regulator of NFAT activation, calcineurin, with the FDA-approved therapeutic FK506 (Tacrolimus) modestly improved neuroinflammation in Ppt1-/- mice. These findings indicate that the absence of depalmitoylation stifles synaptic protein trafficking and contributes to neuroinflammation via an Akap5-associated mechanism.
RESUMEN
The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain's processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABAA receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABAA receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.
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Hormona Liberadora de Corticotropina , Habénula , Animales , Masculino , Ratones , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Endocannabinoides , Habénula/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Excitation-driven entry of Ca(2+) through L-type voltage-gated Ca(2+) channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of Ca(V)1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to Ca(V)1.2 channels by A-kinase anchoring proteins (AKAPs). Ca(V)1.2 channels also undergo negative autoregulation via Ca(2+)-dependent inactivation (CDI), which strongly limits Ca(2+) entry. An abundance of evidence indicates that CDI relies upon binding of Ca(2+)/calmodulin (CaM) to an isoleucine-glutamine motif in the carboxy tail of Ca(V)1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca(2+)/CaM-activated phosphatase calcineurin (CaN) is scaffolded to Ca(V)1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity-mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase's autoinhibitory domain-interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca(2+)/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling.