Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways.

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein-coupled receptors that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1, and activity-regulated cytoskeleton-associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, whereas only intracellular mGlu5 activates protein phosphatase 2 and leads to fragile X mental retardation protein degradation and de novo protein synthesis followed by a protein synthesis-dependent increase in Arc and post-synaptic density protein 95. However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 internalization and chemically induced long-term depression albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

Location and Cell-Type-Specific Bias of Metabotropic Glutamate Receptor, mGlu5, Negative Allosteric Modulators.

Emerging data indicate that G-protein coupled receptor (GPCR) signaling is determined by not only the agonist and a given receptor but also a variety of cell-type-specific factors that can influence a receptor's response. For example, the metabotropic glutamate receptor, mGlu5, which is implicated in a number of neuropsychiatric disorders such as depression, anxiety, and autism, also signals from inside the cell which leads to sustained Ca2+ mobilization versus rapid transient responses. Because mGlu5 is an important drug target, many negative allosteric modulators (NAMs) have been generated to modulate its activity. Here we show that NAMs such as AFQ056, AZD2066, and RG7090 elicit very different end points when tested in postnatal neuronal cultures expressing endogenous mGlu5 receptors. For example, AFQ056 fails to block intracellular mGlu5-mediated Ca2+ increases whereas RG7090 is very effective. These differences are not due to differential receptor levels, since about the same number of mGlu5 receptors are present on neurons from the cortex, hippocampus, and striatum based on pharmacological, biochemical, and molecular data. Moreover, biotinylation studies reveal that more than 90% of the receptor is intracellular in these neurons. Taken together, these data indicate that the tested NAMs exhibit both location-dependent and cell type specific bias for mGlu5-mediated Ca2+ mobilization which may affect clinical outcomes.

Intracellular GPCRs Play Key Roles in Synaptic Plasticity.

The trillions of synaptic connections within the human brain are shaped by experience and neuronal activity, both of which underlie synaptic plasticity and ultimately learning and memory. G protein-coupled receptors (GPCRs) play key roles in synaptic plasticity by strengthening or weakening synapses and/or shaping dendritic spines. While most studies of synaptic plasticity have focused on cell surface receptors and their downstream signaling partners, emerging data point to a critical new role for the very same receptors to signal from inside the cell. Intracellular receptors have been localized to the nucleus, endoplasmic reticulum, lysosome, and mitochondria. From these intracellular positions, such receptors may couple to different signaling systems, display unique desensitization patterns, and/or show distinct patterns of subcellular distribution. Intracellular GPCRs can be activated at the cell surface, endocytosed, and transported to an intracellular site or simply activated in situ by de novo ligand synthesis, diffusion of permeable ligands, or active transport of non-permeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in synaptic plasticity and learning and memory. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools.

GPCR signalling from within the cell.

Traditionally, signal transduction from GPCRs is thought to emanate from the cell surface where receptor interactions with external stimuli can be transformed into a broad range of cellular responses. However, emergent data show that numerous GPCRs are also associated with various intracellular membranes where they may couple to different signalling systems, display unique desensitization patterns and/or exhibit distinct patterns of subcellular distribution. Although many GPCRs can be activated at the cell surface and subsequently endocytosed and transported to a unique intracellular site, other intracellular GPCRs can be activated in situ either via de novo ligand synthesis, diffusion of permeable ligands or active transport of nonpermeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in various biological functions including learning and memory, contractility and angiogenesis. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools. LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization.

Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11 Here, we identified a region within the C-terminal domain (amino acids 852-876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+in situ.

Comprehensive functional characterization of murine infantile Batten disease including Parkinson-like behavior and dopaminergic markers.

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is a neurodegenerative lysosomal storage disease caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). The PPT1-deficient mouse (Cln1(-/-)) is a useful phenocopy of human INCL. Cln1(-/-) mice display retinal dysfunction, seizures, motor deficits, and die at ~8 months of age. However, little is known about the cognitive and behavioral functions of Cln1(-/-) mice during disease progression. In the present study, younger (~1-2 months of age) Cln1(-/-) mice showed minor deficits in motor/sensorimotor functions while older (~5-6 months of age) Cln1(-/-) mice exhibited more severe impairments, including decreased locomotor activity, inferior cued water maze performance, decreased running wheel ability, and altered auditory cue conditioning. Unexpectedly, certain cognitive functions such as some learning and memory capabilities seemed intact in older Cln1(-/-) mice. Younger and older Cln1(-/-) mice presented with walking initiation defects, gait abnormalities, and slowed movements, which are analogous to some symptoms reported in INCL and parkinsonism. However, there was no evidence of alterations in dopaminergic markers in Cln1(-/-) mice. Results from this study demonstrate quantifiable changes in behavioral functions during progression of murine INCL and suggest that Parkinson-like motor/sensorimotor deficits in Cln1(-/-) mice are not mediated by dopamine deficiency.