Limin Mao

The Scaffold Protein Homer1b/c Links Metabotropic Glutamate Receptor 5 to Extracellular Signal-Regulated Protein Kinase Cascades in Neurons

Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP3) and thereby trigger intracellular Ca2+ release. Also, group I mGluRs are organized with members of Homer scaffold proteins into multiprotein complexes involved in postreceptor signaling. In this study, we investigated the relative importance of the IP3/Ca2+ signaling and novel Homer proteins in group I mGluR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured rat striatal neurons. We found that selective activation of mGluR5, but not mGluR1, increased ERK1/2 phosphorylation. Whereas the IP3/Ca2+ cascade transmits a small portion of signals from mGluR5 to ERK1/2, the member of Homer family Homer1b/c forms a central signaling pathway linking mGluR5 to ERK1/2 in a Ca2+-independent manner. This was demonstrated by the findings that the mGluR5-mediated ERK1/2 phosphorylation was mostly reduced by a cell-permeable Tat-fusion peptide that selectively disrupted the interaction of mGluR5 with the Homer1b/c and by small interfering RNAs that selectively knocked down cellular levels of Homer1b/c proteins. Furthermore, ERK1/2, when only coactivated by both IP3/Ca2+- and Homer1b/c-dependent pathways, showed the ability to phosphorylate two transcription factors, Elk-1 and cAMP response element-binding protein, and thereby facilitated c-Fos expression. Together, we have identified two coordinated signaling pathways (a conventional IP3/Ca2+ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca2+-dependent and -independent pathways are corequired to activate ERK1/2 to a level sufficient to achieve the mGluR5-dependent synapse-to-nucleus communication imperative for the transcriptional regulation.

Regulation of mitogen‐activated protein kinases by glutamate receptors

Glutamate receptors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors within the nucleus. The mitogen‐activated protein kinase (MAPK) cascade is one of the best characterized cascades in this regulatory process. The Ca2+‐permeable ionotropic glutamate receptor, mainly the NMDA receptor subtype, activates MAPKs through a biochemical route involving the Ca2+‐sensitive Ras‐guanine nucleotide releasing factor, Ca2+/calmodulin‐dependent protein kinase II, and phosphoinositide 3‐kinase. The metabotropic glutamate receptor (mGluR), however, activates MAPKs primarily through a Ca2+‐insensitve pathway involving the transactivation of receptor tyrosine kinases. The adaptor protein Homer also plays a role in this process. As an information superhighway between surface glutamate receptors and transcription factors in the nucleus, active MAPKs phosphorylate specific transcription factors (Elk‐1 and CREB), and thereby regulate distinct programs of gene expression. The regulated gene expression contributes to the development of multiple forms of synaptic plasticity related to long‐lasting changes in memory function and addictive properties of drugs of abuse. This review, by focusing on new data from recent years, discusses the signaling mechanisms by which different types of glutamate receptors activate MAPKs, features of each MAPK cascade in regulating gene expression, and the importance of glutamate/MAPK‐dependent synaptic plasticity in memory and addiction.

A Novel Ca2+-Independent Signaling Pathway to Extracellular Signal-Regulated Protein Kinase by Coactivation of NMDA Receptors and Metabotropic Glutamate Receptor 5 in Neurons

The specification and organization of glutamatergic synaptic transmission require the coordinated interaction among glutamate receptors and their synaptic adaptor proteins closely assembled in the postsynaptic density (PSD). Here we investigated the interaction between NMDA receptors and metabotropic glutamate receptor 5 (mGluR5) in the integral regulation of extracellular signal-regulated protein kinase (ERK) and gene expression in cultured rat striatal neurons. We found that coapplication of NMDA and the mGluR5 agonist (S)-3,5-dihydroxyphenylglycine synergistically increased ERK phosphorylation. Interestingly, the synergistic increase in ERK phosphorylation was dependent on the cross talk between NMDA receptor-associated synaptic adaptor protein PSD-95 and the mGluR5-linked adaptor protein Homer1b/c but not on the conventional Ca2+ signaling derived from NMDA receptors (Ca2+ influx) and mGluR5 (intracellular Ca2+ release). This was demonstrated by the findings that the synergistic phosphorylation of ERK induced by coactivation of NMDA receptors and mGluR5 was blocked by either a Tat peptide that disrupts NMDA receptor/PSD-95 binding or small interfering RNAs that selectively reduce cellular levels of Homer1b/c. Furthermore, ERK activated through this PSD-95/Homer1b/c-dependent and Ca2+-independent pathway was able to phosphorylate the two key transcription factors Elk-1 and cAMP response element-binding protein, which further leads to facilitation of c-Fos expression. Together, we have identified a novel Ca2+-independent signaling pathway to ERK by the synergistic interaction of NMDA receptors and mGluR5 via their adaptor proteins in the PSD of neurons, which underlies a synapse-to-nucleus communication important for the transcriptional regulation.

Phosphorylation of AMPA receptors: mechanisms and synaptic plasticity.

The ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.

Activity-Dependent Modulation of Limbic Dopamine D3 Receptors by CaMKII

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is central to synaptic transmission. Here we show that synaptic CaMKIIalpha binds to the N-terminal region of the third intracellular loop of the limbic dopamine D3 receptor (D3R). This binding is Ca(2+) sensitive and is sustained by autophosphorylation of CaMKII, providing an unrecognized route for the Ca(2+)-mediated regulation of D3Rs. The interaction of CaMKIIalpha with D3Rs transforms D3Rs into a biochemical substrate of the kinase and promotes the kinase to phosphorylate D3Rs at a selective serine site (S229). In accumbal neurons in vivo, CaMKIIalpha is recruited to D3Rs by rising Ca(2+) to increase the CaMKIIalpha-mediated phosphorylation of D3Rs, thereby transiently inhibiting D3R efficacy. Notably, the D3R inhibition is critical for integrating dopamine signaling to control behavioral sensitivity to the psychostimulant cocaine. Our data identify CaMKIIalpha as a recruitable regulator of dopamine receptor function. By binding and phosphorylating limbic D3Rs, CaMKIIalpha modulates dopamine signaling and psychomotor function in an activity-dependent manner.

Comparison of the antinociceptive effects induced by electroacupuncture and transcutaneous electrical nerve stimulation in the rat

The analgesic effects induced by two different kinds of peripheral conditioning stimulations, electroacupuncture (EA) and transcutaneous electrical nerve stimulation (TENS), were compared in the rat using the latency of radiant heat-evoked tail flick reflex as nociceptive index. The parallel elevations of withdrawal latency of tail flick were produced by EA and TENS administrations at the acupoints of S36 and Sp6 with low intensity (1-2-3 mA) and one of three different frequencies (2, 15 and 100 Hz). Analgesic effects of EA or TENS were characterized by slow-on and slow-off nature, and a significant linear correlation was found between both at any one of three frequencies. Systemic naloxone hydrochloride (2 mg/kg) almost completely and partially antagonized 2 and 15 Hz EA- or TENS-induced analgesia, respectively, but failed to affect those induced by 100 Hz EA or TENS. Tolerance to EA stimulation with one of three frequencies reduced the corresponding frequency TENS-induced analgesia and vice versa. These data indicate that: (a) there is no significant difference in producing antinociception for two different peripheral conditioning stimulations when applied at the same sites and (b) the common neural mechanisms most likely process the analgesic effects of EA and TENS. The involvement of (an) endogenous opiate mechanism in the management of different frequency EA and TENS analgesia is discussed in detail.

Glutamate signaling to Ras-MAPK in striatal neurons: mechanisms for inducible gene expression and plasticity.

Extracellular signals can regulate mitogen-activated protein kinase (MAPK) cascades through a receptor-mediated mechanism in postmitotic neurons of adult mammalian brain. Both ionotropic and metabotropic glutamate receptors (mGluRs) are found to possess such an ability in striatal neurons. NMDA and AMPA receptor signals seem to share a largely common route to MAPK phosphorylation which involves first activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ influx, followed by subsequent induction of phosphoinositide 3-kinase (PI3-kinase). Through its lipid and protein kinase activity, active PI3-kinase may transduce signals to Ras-MAPK cascades via at least two distinct pathways. A novel, Ca2+-independent pathway is believed to mediate mGluR signals to Ras-MAPK activation. As an information superhighway between the surface membrane and the nucleus, Ras-MAPK cascades, through activating their specific nuclear transcription factor targets, are actively involved in the regulation of gene expression. Emerging evidence shows that MAPK-mediated genomic responses in striatal neurons to drug exposure contribute to the development of neuroplasticity related to addictive properties of drugs of abuse.

Profound astrogenesis in the striatum of adult mice following nigrostriatal dopaminergic lesion by repeated MPTP administration.

Neural progenitor cells are present in the rodent brain throughout adulthood, and can proliferate and differentiate into new neurons and/or glia to repair injury. To explore the repair processes mediated by brain progenitor cells, a selective lesion of the nigrostriatal dopaminergic pathway was induced in young adult mice by repeated administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). A thymidine analog, bromodeoxyuridine (BrdU), was used as a tracer for DNA synthesis to label the dividing cells and their terminal progeny following injury. Three days after MPTP treatments (25 mg/kg, once daily for 5 days), an 8-fold increase in the number of BrdU-labeled newborn cells was observed in the dorsal striatum. A 5-fold increase was also seen in the substantia nigra (SN). Newborn cells in the striatum survived beyond 60 days after their birth whereas newborn cells in the SN survived for less than 31 days. The vast majority of newborn cells in the striatum differentiated into astroglia according to their radial morphology and co-expression with an astroglial marker, S100beta, within 10 days after birth. In contrast, most BrdU-positive cells in the SN failed to co-express S100beta. Little or none of BrdU-labeled cells in both the striatum and SN were found to co-localize with a neuronal marker, neuronal nuclear antigen, or tyrosine hydroxylase during the full course of survival days surveyed (3 to 60 days). Repeated MPTP also decreased dopamine content and uptake in the striatum, which showed a significant recovery 31 days after MPTP lesion. These results demonstrate a rapid and profound astrogenesis in the striatum of young adult mice in response to toxic dopaminergic insult. The lack of neurogenesis in the two affected brain areas indicates the relative importance of glial cell regeneration in repairing MPTP injury.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Gαq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

The arcuate nucleus of hypothalamus mediates low but not high frequency electroacupuncture analgesia in rats

Electrolytic, kainic acid or sham lesions were made in the arcuate nucleus of the hypothalamus (ARH) in female Wistar rats to investigate the putative role of the ARH in the organization of low (2 Hz) or high (100 Hz) frequency electroacupuncture (EA) analgesia. Both electrolytic and chemical lesions lead to an almost total suppression of the low frequency EA analgesia as measured 4 and 6 days following the surgical intervention, leaving high frequency EA analgesia unaffected. In sham-operated animals, the antinociceptive effect induced by low or high frequency EA was essentially intact. These data indicate that neurones of the ARH most likely play an important role in mediating low, but not high frequency EA analgesia.