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.

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. To address this issue, we used cellular, optical and molecular techniques to show that intracellular glutamate is largely sequestered in mitochondria; that the glutamate concentration necessary to activate intracellular mGluR5 is about ten-fold higher than what is necessary to activate cell surface mGluR5; and uncaging caged glutamate within neurons can directly activate the receptor. Thus these studies further the concept that glutamate itself serves as the ligand for intracellular mGluR5.

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. Nerve injury inducing nociceptive hypersensitivity also increases the expression of nuclear mGluR5 and receptor-mediated phosphorylated-ERK1/2, Arc/Arg3.1 and c-fos. Spinal blockade of intracellular mGluR5 reduces neuropathic pain behaviours and signalling molecules, whereas blockade of cell-surface mGluR5 has little effect. Decreasing intracellular glutamate via blocking EAAT-3, mimics the effects of intracellular mGluR5 antagonism. These findings show a direct link between an intracellular GPCR and behavioural expression in vivo. Blockade of intracellular mGluR5 represents a new strategy for the development of effective therapies for persistent pain.

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.

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.

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. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells.

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., the particular membrane and/or cell type from which it signals). Because mGlu5 receptors play important roles in both normal development and in disorders such as Fragile X syndrome, autism, epilepsy, addiction, anxiety, schizophrenia, pain, dyskinesias, and melanoma, a large number of drugs are being developed to allosterically target this receptor. Therefore, it is critical to understand how such drugs might be affecting mGlu5 receptor function on different membranes and in different brain regions. Further elucidation of the site(s) of action of these drugs may determine which signal pathways mediate therapeutic efficacy.

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. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment.

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., 2012). To determine whether mGluR5 signals from intracellular membranes of other cell types, such as excitatory pyramidal neurons in the hippocampus, we used dissociated rat CA1 hippocampal cultures and slice preparations to localize and characterize endogenous receptors. As in the striatum, CA1 neurons exhibited an abundance of mGluR5 both on the cell surface and intracellular membranes, including the endoplasmic reticulum and the nucleus where it colocalized with the sodium-dependent excitatory amino acid transporter, EAAT3. Inhibition of EAAT3 or sodium-free buffer conditions prevented accumulations of radiolabeled agonist. Using a pharmacological approach to isolate different pools of mGluR5, both intracellular and cell surface receptors induced oscillatory Ca(2+) responses in dissociated CA1 neurons; however, only intracellular mGluR5 activation triggered sustained high amplitude Ca(2+) rises in dendrites. Consistent with the notion that mGluR5 can signal from intracellular membranes, uncaging glutamate on a CA1 dendrite led to a local Ca(2+) rise, even in the presence of ionotropic and cell surface metabotropic receptor inhibitors. Finally, activation of intracellular mGluR5 alone mediated both electrically induced and chemically induced long-term depression, but not long-term potentiation, in acute hippocampal slices. These data suggest a physiologically relevant and important role for intracellular mGluR5 in hippocampal synaptic plasticity.

MPP+-induces PUMA- and p53-dependent, but ATF3-independent cell death.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and depletion of striatal dopamine (DA), leading to a range of motor symptoms, including resting tremor, rigidity, bradykinesia and postural abnormalities. The neurotoxin (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinium (MPP(+)), cause dopaminergic cell loss in a variety of animal species and produce symptoms similar to those seen in PD. Our lab has shown that MPP(+) activates cell stress pathways, including the unfolded protein response (UPR) in mouse primary mesencephalic cultures. The BH3-only protein, PUMA (p53 upregulated mediator of apoptosis), has been shown to be activated in response to many cellular stresses, including endoplasmic reticulum (ER) stress and UPR, and to induce cell death. Therefore, we hypothesized that PUMA may mediate MPP(+) toxicity. To test this hypothesis, we compared the response of primary mesencephalic cultures from wild-type and PUMA deficient (-/-) mice to MPP(+). We also utilized cultures from p53 -/- and activating transcription factor 3 (ATF3) -/- mice to further elucidate the pathways involved. These studies revealed that PUMA and p53, but not ATF3, are required for MPP(+)-induced cell death, suggesting that UPR activation is parallel to the induction of MPP(+)-induced cell death.

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. To determine whether LRRK2 contains a region of protein responsible for self-aggregation, two independent, bioinformatic algorithms were used to identify an N-terminal amino acid sequence as being aggregation-prone. Cells subsequently transfected with a construct containing this domain were found to have significantly increased protein aggregation compared to wild type protein or a construct containing only the last half of the molecule. Finally, in support of the hypothesis that aggregates represent a self-protection strategy, aggregated N-terminal LRRK2 constructs significantly attenuated cell death induced by the PD-mimetic, 6-hydroxydopamine (6-OHDA).

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. The device comprises two large open culture chambers connected by a parallel array of microchannels that achieves fluidic separation of axons from the soma and allows the tracking of mitochondrial movement along oriented axons. Dopaminergic neurons from midbrain cultures were successfully cultured within the microdevices for up to 4 weeks and extended their axons across the microchannels. Axonal mitochondria within the microchannels were labeled by transduction with a mitochondrial-targeted DsRed2 lentiviral vector or with the mitochondria-specific dye, Mitotracker Deep Red and were visually tracked with conventional confocal microscopy. The methodology and device that we have described here will allow further study of the role of mitochondrial transport defects play in major neurodegenerative diseases.

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. RESULTS: Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that WldS but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive WldS still protects. In addition, NAD+ by itself is axonally protective and together with WldS is additive in the MPP+ model. CONCLUSIONS: Our data suggest that NAD+ and WldS act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that WldS might be involved in preserving mitochondrial health or maintaining cellular metabolism.

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., Kumar, V., and O'Malley, K. L. (2009) J. Biol. Chem. 284, 35827-35838). Using pharmacological, genetic, and bioinformatics approaches, the current findings show that both receptor populations up-regulate many immediate early genes involved in growth and differentiation. Activation of intracellular mGluR5 also up-regulates genes involved in synaptic plasticity including activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mechanistically, intracellular mGluR5-mediated Arc induction is dependent upon extracellular and intracellular Ca(2+) and ERK1/2 as well as calmodulin-dependent kinases as known chelators, inhibitors, and a dominant negative Ca(2+)/calmodulin-dependent protein kinase II construct block Arc increases. Moreover, intracellular mGluR5-induced Arc expression requires the serum response transcription factor (SRF) as wild type but not SRF-deficient neurons show this response. Finally, increased Arc levels due to high K(+) depolarization is significantly reduced in response to a permeable but not an impermeable mGluR5 antagonist. Taken together, these data highlight the importance of intracellular mGluR5 in the cascade of events associated with sustained synaptic transmission.

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+). MPP+ rapidly reduced overall mitochondrial motility in DA axons; among motile mitochondria, anterograde transport was slower yet retrograde transport was increased. Transport effects were specific for DA mitochondria, which were smaller and transported more slowly than their non-DA counterparts. MPP+ did not affect synaptophysin-tagged vesicles or any other measureable moving particle. Toxin effects on DA mitochondria were not dependent upon ATP, calcium, free radical species, JNK, or caspase3/PKC pathways but were completely blocked by the thiol-anti-oxidant N-acetyl-cysteine or membrane-permeable glutathione. Since these drugs also rescued processes from degeneration, these findings emphasize the need to develop therapeutics aimed at axons as well as cell bodies to preserve "normal" circuitry and function as long as possible.

6-OHDA generated ROS induces DNA damage and p53- and PUMA-dependent cell death.

BACKGROUND: Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN), resulting in tremor, rigidity, and bradykinesia. Although the etiology is unknown, insight into the disease process comes from the dopamine (DA) derivative, 6-hydroxydopamine (6-OHDA), which produces PD-like symptoms. Studies show that 6-OHDA activates stress pathways, such as the unfolded protein response (UPR), triggers mitochondrial release of cytochrome-c, and activates caspases, such as caspase-3. Because the BH3-only protein, Puma (p53-upregulated mediator of apoptosis), is activated in response to UPR, it is thought to be a link between cell stress and apoptosis. RESULTS: To test the hypothesis that Puma serves such a role in 6-OHDA-mediated cell death, we compared the response of dopaminergic neurons from wild-type and Puma-null mice to 6-OHDA. Results indicate that Puma is required for 6-OHDA-induced cell death, in primary dissociated midbrain cultures as well as in vivo. In these cultures, 6-OHDA-induced DNA damage and p53 were required for 6-OHDA-induced cell death. In contrast, while 6-OHDA led to upregulation of UPR markers, loss of ATF3 did not protect against 6-OHDA. CONCLUSIONS: Together, our results indicate that 6-OHDA-induced upregulation of Puma and cell death are independent of UPR. Instead, p53 and DNA damage repair pathways mediate 6-OHDA-induced toxicity.

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. Specifically, Nf1 OPG mice exhibit reduced rearing in response to novel objects and environmental stimuli. Similar to children with NF1, the attention system dysfunction in these mice is reversed by treatment with methylphenidate (MPH), suggesting a defect in brain catecholamine homeostasis. We further demonstrate that this attention system abnormality is the consequence of reduced dopamine (DA) levels in the striatum, which is normalized following either MPH or l-dopa administration. The reduction in striatal DA levels in Nf1 OPG mice is associated with reduced striatal expression of tyrosine hydroxylase, the rate-limited enzyme in DA synthesis, without any associated dopaminergic cell loss in the substantia nigra. Moreover, we demonstrate a cell-autonomous defect in Nf1+/- dopaminergic neuron growth cone areas and neurite extension in vitro, which results in decreased dopaminergic cell projections to the striatum in Nf1 OPG mice in vivo. Collectively, these data establish abnormal DA homeostasis as the primary biochemical defect underlying the attention system dysfunction in Nf1 GEM relevant to children with NF1.