Ubiquilin (UBQL) is a member of type 2 ubiquitin-like (UBL) protein family. They structurally contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated (UBA) domain. Ubiquilin 2 (UBQL2) physically associates with poly ubiquitinated proteins and delivers them to the proteasome for degradation. This protein has been shown to play an important role in the regulation of aggregation and degradation of various neurodegenerative disease-associated proteins. In this study, we looked into the role of the ubiquilin-2 proteins in the AMPA receptor ubiquitination and proteasomal degradation pathway. Our results indicate that UBQL2 overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, by co-immunoprecipitation we found that UBQL2 interacts with ubiquitinated AMPARs. We, therefore propose that UBQL2 brings AMPARs to the proteasome for degradation. Consistent with this notion, expression of UBQL2 P497H, a mutant form incapable of interaction with proteasome, causes accumulation of AMPA receptors. These results indicate a role for UBQL2 in associating with and directing ubiquitinated AMPA receptors to the proteasome for degradation. / 2020-08-09T00:00:00Z

Noradrenergic signaling has important functions in the central nervous system (CNS) with respect to emotion, learning and memory. Activation of β- adrenergic receptors (β ARs) stimulates protein kinase A via Gs-protein, adenylyl cyclase, and cAMP. Synaptic β←2 -adrenergic receptors, targets of the neurotransmitter norephinephrin, are associated with the GluA1 subunit of AMPA-type glutamate receptors, which mediate most excitatory synaptic transmission in mammalian CNS. PKA-mediated phosphorylation of GluA1 on Ser845 is important for GluA1 surface expression, activity induced postsynaptic accumulation, and synaptic plasticity. Postsynaptic localization of PKA is mediated by a major scaffolding protein `A kinase anchor protein 5 (AKAP5)'. AKAP5 associates with AMPA receptors via SAP97 and PSD95. We have two strains of AKAP5 mutant mice: AKAP5 knockout and AKAP5 D36. AKAP5 KO mice have a complete loss of AKAP5 gene expression. D36 mice miss the last 36 residues (PKA binding site) of AKAP5 but without affecting other interactions. These mutant mice provide us with appropriate in vivo models for studying the functional roles of AKAP5. We compared the functional and physical association of β2AR and AMPA receptors among wild type, AKAP5 KO, and AKAP5 D36 mice. Although AKAP5 was not necessary for the assembly of the β2AR / GluA1 complex, we found that AKAP5 anchored PKA activity was required for full β2AR stimulation-induced GluA1 Ser845 phosphorylation. Recording and analysis of field EPSPs (fEPSPs) of CA1 pyramidal neurons with brief bath perfusion of the β2AR agonist isoproterenol indicated a role of AKAP5 anchored PKA in the regulation of postsynaptic AMPAR responses by norephinephrin. Moreover, we observed a delayed extinction of contextual fear memory in AKAP5 D36 mice, which suggests the involvement of AKAP5 anchored PKA in memory formation and modification.

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex.

<p>Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.</p> <p>In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.</p> / Dissertation

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex.

No description available.

The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.

Ketamine is a non-competitive NMDA antagonist that has been shown to have antidepressant properties both clinically as well as in preclinical studies when administered at a subanesthetic dose. In vivo electrophysiological recordings were carried in male Sprague Dawley rats 30 minutes following ketamine administration (10 mg/kg) to first assess its effects on monoaminergic firing. Whilst no change in the firing activity of serotonin (5-HT) neurons was observed in the dorsal raphe nucleus (DRN), an increase in the firing activity was observed for dopamine (DA) and noradrenergic (NE) neurons in the ventral tegmental area (VTA) and locus coeruleus (LC), respectively. The effect of ketamine on these electrophysiological parameters was prevented by pre-administration of the AMPA receptor antagonist NBQX 10 minutes prior to ketamine administration. In a second series of experiments, an increase in AMPA-evoked response was observed within 30 minutes in the CA3 layer of the hippocampus (HPC) following acute ketamine administration. These findings suggest that acute ketamine administration produces a prompt enhancement of AMPA transmission in the forebrain and also results in increased catecholaminergic activity. These effects may play a crucial role in the rapid antidepressant effects of ketamine observed shortly following its infusion in the clinic.

Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate CNS. In many biological systems there are changes in the properties of AMPARs during development that are essential for providing an increase in efficiency of information transfer between neurons and a refinement of motor co-ordination and sensory perception and cognition. It is not surprising that improper development or loss of function of AMPARs can lead to many neurological disorders such as epilepsy and amyotrophic lateral sclerosis. Thus, determining the mechanisms by which AMPARs mature is of particular importance. The objectives of my thesis were to characterize the developmental changes in AMPAR-mediated currents in zebrafish Mauthner cells and to determine the mechanisms underlying any changes. The major findings reported in this thesis are that (1) there are developmental changes in the properties of AMPAR-currents as the Mauthner cell matures; (2) the mechanism underlying these changes is a switch in the composition of AMPA receptor subtypes; and (3) PKC is necessary for the developmental switch in AMPAR subtypes from slow receptors to fast receptors. These findings provide valuable insights into the mechanism underlying the development of AMPARs. In addition, they provide the first instance of a signalling link (PKC) required for the developmental subunit switch and the developmental speeding of AMPAR kinetics. / Physiology, Cell Biology and Developmental Biology

Thesis (Ph.D. in Neuroscience) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 116-128). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;