Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95

The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.     

Synaptic drosophila UNC‐13 is regulated by antagonistic G‐protein pathways via a proteasome‐dependent degradation mechanism

UNC‐13 is a highly conserved plasma membrane‐associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC‐13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion (“priming”) at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC‐13 in NMJ synaptic boutons is regulated downstream of GαS and Gαq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC‐13 synaptic up‐regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC‐13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC‐13, through a mechanism that appears independent of PKC. Our results suggest that proteasome‐mediated protein degradation is the primary mechanism regulating DUNC‐13 levels at the synapse. Both PLC‐ and PKA‐mediated pathways appear to regulate synaptic levels of DUNC‐13 through controlling the rate of proteasome‐dependent DUNC‐13 degradation. We conclude that the functional abundance of DUNC‐13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 417–438, 2003     

Glutamate receptor dynamics in dendritic microdomains

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.     

Intracellular signaling mechanisms that shape postsynaptic GABAergic synapses

Postsynaptic GABAergic receptors interact with various membrane and intracellular proteins to mediate inhibitory synaptic transmission. They form structural and/or signaling synaptic protein complexes that perform a variety of postsynaptic functions. In particular, the key GABAergic synaptic scaffold, gephyrin, and its interacting partners govern downstream signaling pathways that are essential for GABAergic synapse development, transmission, and plasticity. In this review, we discuss recent researches on GABAergic synaptic signaling pathways. We also outline the main outstanding issues that need to be addressed in this field and highlight the association of dysregulated GABAergic synaptic signaling with the onset of various brain disorders.     

Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity

Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.     

Lipid rafts in neuregulin signaling at synapses

Neuregulins are a family of EGF domain-containing factors that play an important role in development. In the nervous system, they promote glial differentiation, induce neurotransmitter receptor expression, and regulate synaptic plasticity. Recent studies indicate that ErbB protein tyrosine kinases, neuregulin receptors, translocate to lipid raft microdomains in the plasma membrane in response to neuregulin. Localization of ErbB proteins in lipid rafts appeared to be necessary for neuregulin signaling and regulation of synaptic plasticity. We will review recent studies of lipid rafts and neuregulin function and discuss possible roles of lipid rafts in compartmentalized neuregulin signaling and translocation of ErbB proteins to synapses.     

Protein kinase C activity and isoform expression during early postnatal development of rat myocardium

Total protein kinase C (PKC) activity, its isoform expression, and concentration and fatty acid (FA) composition of diacylglycerol (DAG) were determined in the left ventricular myocardium of the rat during early postnatal development (d 2, 3, 5, 7, and 10). PKC activity measured by the incorporation of ³²P into histone IIIS decreased between d 2 and 10 in the homogenate as well as in cytosolic, membrane (100,000g), and nuclear-cytoskeletal-myofilament fractions (1000g). Likewise, the expression of PKC isoforms (α, δ, and ε) determined by immunoblotting generally declined during the period analyzed, although with a variable pattern. In the membrane and nuclear cytoskeletal myofilament fractions, PKCδ and PKCε expression decreased markedly by d 3, returning to or close to the d 2 level immediately on d 5. PKCα expression in the membrane fraction remained almost unchanged by d 7, declining thereafter. PKCδ and PKCε were associated predominantly with particulate fractions, whereas PKCα was more abundant in the cytosolic fraction. DAG concentration exhibited a significant decline by d 5, consistent with the decrease in maximal PKC activity. The unsaturation index of FA in DAG tended to decrease on d 3 owing to the lowered proportion of all polyunsaturated FA of n−6 and n−3 series. These results demonstrate that the developmental decrease in PKC activity and expression in the rat myocardium is not linear and that subcellular localization of the enzyme exhibits isoform-specific day-by-day changes during the early postnatal period. These changes are compatible with the view that PKC signaling may be involved in the control of a rapid switch of myocardial growth pattern during the first week of life.     

Homeostatic synaptic plasticity: from single synapses to neural circuits

Homeostatic synaptic plasticity remains an enigmatic form of synaptic plasticity. Increasing interest on the topic has fuelled a surge of recent studies that have identified key molecular players and the signaling pathways involved. However, the new findings also highlight our lack of knowledge concerning some of the basic properties of homeostatic synaptic plasticity. In this review we address how homeostatic mechanisms balance synaptic strengths between the presynaptic and the postsynaptic terminals and across synapses that share the same postsynaptic neuron.     

PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity

Activity-dependent modification of excitatory synaptic transmission is a fundamental mechanism for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the postsynaptic density, a highly organized protein network. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in regulating both basal synaptic transmission and synaptic plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.     

Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.     

Fragile X mental retardation protein in learning-related synaptic plasticity

Fragile X syndrome (FXS) is caused by a lack of the fragile X mental retardation protein (FMRP) due to silencing of the Fmr1 gene. As an RNA binding protein, FMRP is thought to contribute to synaptic plasticity by regulating plasticity-related protein synthesis and other signaling pathways. Previous studies have mostly focused on the roles of FMRP within the hippocampus - a key structure for spatial memory. However, recent studies indicate that FMRP may have a more general contribution to brain functions, including synaptic plasticity and modulation within the prefrontal cortex. In this brief review, we will focus on recent studies reported in the prefrontal cortex, including the anterior cingulate cortex (ACC). We hypothesize that alterations in ACC-related plasticity and synaptic modulation may contribute to various forms of cognitive deficits associated with FXS.     

ERK and mTOR signaling couple beta-adrenergic receptors to translation initiation machinery to gate induction of protein synthesis-dependent long-term potentiation

beta-Adrenergic receptors critically modulate long-lasting synaptic plasticity and long-term memory in the mammalian hippocampus. Persistent long-term potentiation of synaptic strength requires protein synthesis and has been correlated with some forms of hippocampal long-term memory. However, the intracellular processes that initiate protein synthesis downstream of the beta-adrenergic receptor are unidentified. Here we report that activation of beta-adrenergic receptors recruits ERK and mammalian target of rapamycin signaling to facilitate long-term potentiation maintenance at the level of translation initiation. Treatment of mouse hippocampal slices with a beta-adrenergic receptor agonist results in activation of eukaryotic initiation factor 4E and the eukaryotic initiation factor 4E kinase Mnk1, along with inhibition of the translation repressor 4E-BP. This coordinated activation of translation machinery requires concomitant ERK and mammalian target of rapamycin signaling. Taken together, our data identify distinct signaling pathways that converge to regulate beta-adrenergic receptor-dependent protein synthesis during long-term synaptic potentiation in the hippocampus. We suggest that beta-adrenergic receptors play a crucial role in gating the induction of long-lasting synaptic plasticity at the level of translation initiation, a mechanism that may underlie the ability of these receptors to influence the formation of long-lasting memories.     

Retrolinkin cooperates with endophilin A1 to mediate BDNF-TrkB early endocytic trafficking and signaling from early endosomes

Brain-derived neurotrophic factor (BDNF) binds to its cell surface receptor TrkB to regulate differentiation, development, synaptic plasticity, and functional maintenance of neuronal cells. Binding of BDNF triggers TrkB dimerization and autophosphorylation, which provides docking sites for adaptor proteins to recruit and activate downstream signaling molecules. The molecular mechanisms underlying BDNF-TrkB endocytic trafficking crucial for spatiotemporal control of signaling pathways remain to be elucidated. Here we show that retrolinkin, a transmembrane protein, interacts with endophilin A1 and mediates BDNF-activated TrkB (pTrk) trafficking and signaling in CNS neurons. We find that activated TrkB colocalizes and interacts with the early endosome marker APPL1. Both retrolinkin and endophilin A1 are required for BDNF-induced dendrite development and acute extracellular signal-regulated kinase activation from early endosomes. Suppression of retrolinkin expression not only blocks BDNF-triggered TrkB internalization, but also prevents recruitment of endophilin A1 to pTrk vesicles trafficking through APPL1-positive endosomes. These findings reveal a novel mechanism for BDNF-TrkB to regulate signaling both in time and space through a specific membrane trafficking pathway.     

Synaptic Drosophila UNC-13 is regulated by antagonistic G-protein pathways via a proteasome-dependent degradation mechanism

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion ("priming") at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GalphaS and Galphaq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways.     

The Role of Neurotrophins in Brain Aging: A Perspective in Honor of Regino Perez-Polo

During brain aging and progression of Alzheimer’s disease, the levels of Aβ and proinflammatory cytokines accumulate very early in the pathogenic process prior to any major degenerative changes. Accumulation of these molecules may impair with signal transduction pathways critical for neuronal health. Neurotrophin signaling is a critical mechanism involved in synaptic plasticity, learning and memory and neuronal health. We have recently shown that exposure to low levels of Aβ impairs BDNF trkB signal transduction, suppressing the Ras/ERK, and the PI3-K/Akt pathways but not the PLCγ pathway. As a result, downstream regulation of gene expression and neuronal viability are impaired. Recently, we have found that at least three agents – Aβ, TNFα, Il-1β – suppress TrkB signaling and act via a common and novel mechanism. These factors all regulate the docking proteins (e.g., IRS and Shc) that link the activated Trk receptor to downstream effectors. While this is a novel mechanism underlying regulation of Trk signaling, such a mechanism has been identified for the insulin/IGF-1 receptor in the presence of proinflammatory cytokines and is one of the mechanisms for insulin/IGF-resistance, which is a key risk factor for type II diabetes (1). We suggest that accumulation of AB and proinflammatory cytokines during aging generates in the brain a “neurotrophin resistance” state that places the brain at risk for cognitive decline and dementia.     

Pain Facilitation and Activity-Dependent Plasticity in Pain Modulatory Circuitry: Role of BDNF-TrkB Signaling and NMDA Receptors

Pain modulatory circuitry in the brainstem exhibits considerable synaptic plasticity. The increased peripheral neuronal barrage after injury activates spinal projection neurons that then activate multiple chemical mediators including glutamatergic neurons at the brainstem level, leading to an increased synaptic strength and facilitatory output. It is not surprising that a well-established regulator of synaptic plasticity, brain-derived neurotrophic factor (BDNF), contributes to the mechanisms of descending pain facilitation. After tissue injury, BDNF and TrkB signaling in the brainstem circuitry is rapidly activated. Through the intracellular signaling cascade that involves phospholipase C, inositol trisphosphate, protein kinase C, and nonreceptor protein tyrosine kinases; N-methyl-D-aspartate (NMDA) receptors are phosphorylated, descending facilitatory drive is initiated, and behavioral hyperalgesia follows. The synaptic plasticity observed in the pain pathways shares much similarity with more extensively studied forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD), which typically express NMDA receptor dependency and regulation by trophic factors. However, LTP and LTD are experimental phenomena whose relationship to functional states of learning and memory has been difficult to prove. Although mechanisms of synaptic plasticity in pain pathways have typically not been related to LTP and LTD, pain pathways have an advantage as a model system for synaptic modifications as there are many well-established models of persistent pain with clear measures of the behavioral phenotype. Further studies will elucidate cellular and molecular mechanisms of pain sensitization and further our understanding of principles of central nervous system plasticity and responsiveness to environmental challenge.     

Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.     

Bipolar disorder: involvement of signaling cascades and AMPA receptor trafficking at synapses

There is increasing evidence that severe mood disorders are associated with impairment of structural plasticity and cellular resilience. Cumulative data demonstrate that mood stabilizers regulate intracellular signaling cascades, including protein kinase C (PKC), PKA, mitogen-activated protein (MAP) kinase, glycogen synthase kinase 3-beta (GSK3-beta) and intracellular calcium, which are signaling pathways that regulate synaptic plasticity. In this context, it is noteworthy that a growing body of data indicates that the glutamatergic system, has a major role in neuronal plasticity and cellular resilience, might be involved in the pathophysiology and treatment of mood disorders. AMPA glutamate-receptor trafficking is important in synaptic plasticity and might play crucial roles in maintaining critical neuronal circuits associated with mood. Two clinically effective, structurally dissimilar, antimanic agents, lithium and valproate (VPA), down-regulate synaptic expression of AMPA receptor subunit GluR1 in hippocampus in chronically treated rats. This reduction in synaptic GluR1 by lithium and VPA is due to attenuated phosphorylation of GluR1 at a specific PKA site (residue 845 of GluR1), which is crucial for AMPA receptor insertion. By contrast,imipramine, which can provoke mania, increases synaptic expression of GluR1 in the hippocampus in vivo. Furthermore, there is ample evidence from preclinical and clinical research that the glutamatergic system is involved in the pathophysiology of mood disorders and that many of the somatic treatments used for mood disorders including antidepressants, mood stabilizers, atypical antipsychotic drugs and electroconvulsive therapy have both direct and indirect effects on the glutamatergic system. Given these findings, further research with medications that specifically affect the glutamatergic system is warranted. Recent studies in our lab have shown that riluzole, a FDA approved medicine that regulates the glutamatergic system, shows antidepressant efficacy in unipolar and bipolar depression. These studies indicate that regulation of glutamate-mediated synaptic plasticity might play a role in the treatment of mood disorders, and raise new avenues for novel therapies for this devastating illness.     

The Onset of Brain Injury and Neurodegeneration Triggers the Synthesis of Docosanoid Neuroprotective Signaling

Bioactive lipid messengers are formed through phospholipase-mediated cleavage of specific phospholipids from membrane reservoirs. Effectors that activate the synthesis of lipid messengers, include ion channels, neurotransmitters, membrane depolarization, cytokines, and neurotrophic factors. In turn, lipid messengers regulate and interact with multiple pathways, participating in the development, differentiation, function (e.g., long-term potentiation and memory), protection, and repair of cells of the nervous system. Overall, bioactive lipids participate in the regulation of synaptic function and dysfunction. Platelet-activating factor (PAF) and COX-2-synthesized PGE₂ modulate synaptic plasticity and memory. Oxidative stress disrupts lipid signaling, fosters lipid peroxidation, and initiates and propagates neurodegeneration. Lipid messengers participate in the interactions among neurons, astrocytes, oligodendrocytes, microglia, cells of the microvasculature, and other cells. A conglomerate of interrelated cells comprises the neurovascular unit. Signaling at the neurovascular unit is clearly altered in the early stages of cerebrovascular disease as well as in neurodegenerations. Here we will provide examples of how signaling by lipids regulates critical events essential for neuronal survival. We will highlight a newly identified, DHA-derived messenger, neuroprotectin D1, which attenuates oxidative stress-induced apoptosis. The specificity and potency of this novel docosanoid (neuroprotectin D1) indicate a potentially important target for therapeutic intervention.