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

Metabotropic glutamate receptor 5 (mGlu) 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 mGlu 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 mGlu as well as the intracellular receptor in a well-known mGlu synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu in striatal cultures and slice preparations.

Location and Cell-Type-Specific Bias of Metabotropic Glutamate Receptor, mGlu, 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, mGlu, 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 Ca mobilization versus rapid transient responses. Because mGlu is an important drug target, many negative allosteric modulators (NAMs) have been generated to modulate its activity.

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.

GPCR signalling from within the cell.

Unlabelled: 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.

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 Ca changes in the nucleoplasm by coupling with G 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.

Mechanisms Associated with Activation of Intracellular Metabotropic Glutamate Receptor, mGluR5.

The group 1 metabotropic glutamate receptor, mGluR5, is found on the cell surface as well as on intracellular membranes where it can mediate both overlapping and unique signaling effects. Previously we have shown that glutamate activates intracellular mGluR5 by entry through sodium-dependent transporters and/or cystine glutamate exchangers. Calibrated antibody labelling suggests that the glutamate concentration within neurons is quite high (~10 mM) raising the question as to whether intracellular mGluR5 is maximally activated at all times or whether a different ligand might be responsible for receptor activation.

Intracellular mGluR5 plays a critical role in neuropathic pain.

Spinal mGluR5 is a key mediator of neuroplasticity underlying persistent pain. Although brain mGluR5 is localized on cell surface and intracellular membranes, neither the presence nor physiological role of spinal intracellular mGluR5 is established. Here we show that in spinal dorsal horn neurons >80% of mGluR5 is intracellular, of which ∼60% is located on nuclear membranes, where activation leads to sustained Ca(2+) responses.

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.

Reassessment of the role of aromatic amino acid hydroxylases and the effect of infection by Toxoplasma gondii on host dopamine.

Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo.

Location-dependent signaling of the group 1 metabotropic glutamate receptor mGlu5.

Although G protein-coupled receptors are primarily known for converting extracellular signals into intracellular responses, some receptors, such as the group 1 metabotropic glutamate receptor, mGlu5, are also localized on intracellular membranes where they can mediate both overlapping and unique signaling effects. Thus, besides "ligand bias," whereby a receptor's signaling modality can shift from G protein dependence to independence, canonical mGlu5 receptor signaling can also be influenced by "location bias" (i.e.

Functional G protein-coupled receptors on nuclei from brain and primary cultured neurons.

A growing number of G protein-coupled receptors (GPCRs) have been identified on nuclear membranes. In many cases, it is unknown how the intracellular GPCR is activated, how it is trafficked to nuclear membranes, and what long-term signaling consequences follow nuclear receptor activation. Here we describe how to isolate nuclei that are free from plasma membrane and cytoplasmic contamination yet still exhibit physiological properties following receptor activation.

The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons.

6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons.

Intracellular mGluR5 can mediate synaptic plasticity in the hippocampus.

Metabotropic glutamate receptor 5 (mGluR5) is widely expressed throughout the CNS and participates in regulating neuronal function and synaptic transmission. Recent work in the striatum led to the groundbreaking discovery that intracellular mGluR5 activation drives unique signaling pathways, including upregulation of ERK1/2, Elk-1 (Jong et al., 2009) and Arc (Kumar et al.

Sequences located within the N-terminus of the PD-linked LRRK2 lead to increased aggregation and attenuation of 6-hydroxydopamine-induced cell death.

Clinical symptoms of Parkinson's disease (PD) arise from the loss of substantia nigra neurons resulting in bradykinesia, rigidity, and tremor. Intracellular protein aggregates are a pathological hallmark of PD, but whether aggregates contribute to disease progression or represent a protective mechanism remains unknown. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to PD in both familial cases and idiopathic cases and aggregates of the LRRK2 protein are present in postmortem PD brain samples.

A microdevice platform for visualizing mitochondrial transport in aligned dopaminergic axons.

Experimental evidence points to the importance of mitochondrial transport defects in contributing to major neurodegenerative diseases, such as Parkinson's disease (PD). Studies of mitochondrial transport along single axons are difficult with traditional dissociated culture systems and the fragility of the midbrain dopaminergic cultures precludes their survival in previously developed microfluidic devices with an enclosed architecture. Using soft lithography, we generated a microdevice from polydimethylsiloxane (PDMS) for the purpose of studying the transport of mitochondria along single dopaminergic axons.

WldS but not Nmnat1 protects dopaminergic neurites from MPP+ neurotoxicity.

Background: The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury.

Activation of intracellular metabotropic glutamate receptor 5 in striatal neurons leads to up-regulation of genes associated with sustained synaptic transmission including Arc/Arg3.1 protein.

The G-protein coupled receptor, metabotropic glutamate receptor 5 (mGluR5), is expressed on both cell surface and intracellular membranes in striatal neurons. Using pharmacological tools to differentiate membrane responses, we previously demonstrated that cell surface mGluR5 triggers rapid, transient cytoplasmic Ca(2+) rises, resulting in c-Jun N-terminal kinase, Ca(2+)/calmodulin-dependent protein kinase, and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein (CREB) phosphorylation, whereas stimulation of intracellular mGluR5 induces long, sustained Ca(2+) responses leading to the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Elk-1 (Jong, Y. J.

The role of axonopathy in Parkinson's disease.

New genetic and environmental studies of Parkinson's disease have revealed early problems in synaptic function and connectivity indicating that axonal impairment may be an important hallmark in this disorder. Since many studies suggest that axonal dysfunction precedes cell body loss, it is critical to target axons with treatments aimed at preserving "connectivity" as well as to develop and verify "biomarkers" with which to assess disease progression and drug efficacy.

The parkinsonian mimetic, MPP+, specifically impairs mitochondrial transport in dopamine axons.

Impaired axonal transport may play a key role in Parkinson's disease. To test this notion, a microchamber system was adapted to segregate axons from cell bodies using green fluorescent protein-labeled mouse dopamine (DA) neurons. Transport was examined in axons challenged with the DA neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+).

Reduced striatal dopamine underlies the attention system dysfunction in neurofibromatosis-1 mutant mice.

Learning and behavioral abnormalities are among the most common clinical problems in children with the neurofibromatosis-1 (NF1) inherited cancer syndrome. Recent studies using Nf1 genetically engineered mice (GEM) have been instructive for partly elucidating the cellular and molecular defects underlying these cognitive deficits; however, no current model has shed light on the more frequently encountered attention system abnormalities seen in children with NF1. Using an Nf1 optic glioma (OPG) GEM model, we report novel defects in non-selective and selective attention without an accompanying hyperactivity phenotype.

Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts.

G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca(2+) signatures leading to unique cellular responses.

Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.

Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation.

Cell stress induced by the parkinsonian mimetic, 6-hydroxydopamine, is concurrent with oxidation of the chaperone, ERp57, and aggresome formation.

Parkinson's disease (PD) involves an irreversible degeneration of the nigrostriatal pathway. As most cases of PD are sporadic, environmental risk factors may underlie neurodegeneration in dopaminergic neurons. One such factor is 6-hydroxydopamine (6-OHDA), which is widely used as a parkinsonian mimetic.