Regulation of Neuronal Function by Ras-GRF ... - SAGE Journals

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Regulation of Neuronal Function by Ras-GRF Exchange Factors

Genes & Cancer 2(3) 306–319 © The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1947601911408077 http://ganc.sagepub.com

Larry A. Feig

Abstract Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of guanine nucleotide exchange factors (GEFs). The main isoforms, p140-GRF1 and p135-GRF2, have 2 GEF domains that give them the capacity to activate both Ras and Rac GTPases in response to signals from a variety of neurotransmitter receptors. GRF1 and GRF2 proteins are found predominantly in adult neurons of the central nervous system, although they can also be detected in a limited number of other tissues. p140-GRF1 and p135-GRF2 contain calcium/calmodulin-binding IQ domains that allow them to act as calcium sensors to mediate the actions of NMDA-type and calcium-permeable AMPA-type glutamate receptors. p140-GRF1 also mediates the action of dopamine receptors that signal through cAMP. Although p140-GRF1 and p135-GRF2 have similar functional domains, studies of GRF knockout mice show that they can play strikingly different roles in regulating MAP kinase family members, neuronal synaptic plasticity, specific forms of learning and memory, and behavioral responses to psychoactive drugs. In addition, the function of GRF proteins may vary in different regions of the brain. Alternative splice variants yielding smaller GRF1 gene isoforms with fewer functional domains also exist; however, their distinct roles in neurons have not been revealed. Continuing studies of these proteins should yield important insights into the biochemical basis of brain function as well as novel concepts to explain how complex signal transduction proteins, like Ras-GRFs, integrate multiple upstream signals into specific downstream outputs to control brain function. Keywords: Ras-GRF, synaptic plasticity, Ras, neurons

Introduction Ras family GTPases regulate a wide variety of cellular processes including cell proliferation, cell differentiation, and an array of differentiated functions unique to specific cell types. They transmit extracellular signals to specific intracellular signaling cascades by switching from an inactive GDP-bound state to an active GTP-bound state. They then bind to and alter the activity of a set of “effector” proteins (see review1). The activation state of Ras proteins is influenced by 2 types of regulator proteins. Guanine nucleotide exchange factors (GEFs) activate Ras GTPases by promoting the release of bound GDP, thereby allowing activating GTP to take its place (see review2). GTPaseactivating proteins (GAPs), on the other hand, inactivate Ras by enhancing the ability of Ras proteins to hydrolyze bound GTP to GDP (see review3). Multiple GEF families exist to allow Ras activation by multiple extracellular signals. These include the Sos proteins (Sos1 and Sos2) that couple tyrosine kinases to Ras proteins and RasGRP

proteins (GRP1-4) that link G protein– coupled receptors that function through phospholipase C to Ras.4 Finally, and of particular interest in neuronal signaling are the Ras-GRF proteins, 140-kD RasGRF1 (p140-GRF1)5,6 and 130-kD RasGRF2 (p135-GRF2)7 that allow certain neurotransmitter receptors known to increase levels of calcium8,9 or cAMP10 in neurons to activate Ras proteins. The presence of GEFs for Ras in mammalian cells was first detected using biochemical assays on cell lysates.11 Although GRF proteins are now studied mainly in neurons, arguably the most complex of cell types, their discovery and subsequent characterization depended upon genetic studies in much simpler, yeast cells. In those studies, the first exchange factors for Ras, CDC25 and Ste6, were found to be part of regulatory metabolic pathways in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively.12-14 Their properties led to the identification of a mammalian counterpart gene encoding Ras-GRF1 proteins. In 2 studies, 2-kb mouse (referred to as CDC25Mm) and 588-bp

human cDNA clones were isolated by their ability to complement the loss of CDC25 function in yeast or by DNA sequence homology to yeast CDC25.15,16 They encoded 55- and 25-kD fragments of the Ras-activating domains, respectively. At the same time, we used DNA sequence similarity to the yeast CDC25 gene to isolate the full-length 6-kb rat cDNA encoding a 140-kD Ras-GRF1 protein that displayed in vitro Ras-GEF activity.5 The mouse p140 Ras-GRF1 cDNA was subsequently cloned using the partial CDC25Mm clone as a probe.17 As described below, the 55-kD smaller form of GRF1 is a splice variant of the fulllength p140 Ras-GRF1. Although at first it was expected that p140-GRF1 would be the ubiquitous exchange factor connecting tyrosine Departments of Biochemistry and Neuroscience, Tufts University School of Medicine, Boston, MA, USA Corresponding Author: Larry A. Feig, Departments of Biochemistry and Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111 Email: [email protected]

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Role of Ras-GRF proteins in brain function / Feig

Figure 1. Functional domains of p140-GRF1 and p135-GRF2. Both proteins have N-terminal pleckstrin homology (PH) domains, coiled-coil (CC), and IQ domains. These are followed by Dbl homology (DH) and PH domains that lead to the activation of the Rac GTPase and its effector proteins. Their C-terminal regions contain Ras-activating CDC25 domains and their associated Ras exchange motifs (REMs). p140-GRF1 differs from p135-GRF2 in that it contains a “neuronal domain” that binds to the 2B subunit of NMDA-type glutamate receptors. In addition, the CDC25 domain of GRF1, but not that of GRF2, has the ability to activate R-Ras in addition to Ras proteins.

kinases to Ras in all cells, it soon became clear that this was not the case. p140GRF1 is expressed most abundantly in mature neurons of the brain and is limited to only a few other tissues, such as the lung and pancreas.5,6,18 The generic GEFs, turned out to be the Sos proteins, which were cloned subsequently by their homology to a Drosophila Ras-GEF, Son of Sevenless.19 p135-GRF2, which is also expressed preferentially (although not exclusively) in the brain, was eventually cloned based on homology to GRF1.7 Another surprise was the finding that p140-GRF1 and p135-GRF2 contain Racactivating DH domains along with their Ras-activating CDC25 domains (Fig. 1).5,20 While I focus on GRF1 and GRF2 functions in the central nervous system (CNS), a recent review has thoroughly summarized GRF functions in other tissues as well, such as the pancreas.21

Gene Organization and Expression Regulation Paternal Imprinting of Ras-GRF1

A distinguishing feature of the gene encoding GRF1, but not GRF2, is that it is paternally imprinted in such a way

that it is expressed only from the paternal gene in young animals in the brain.22-26 Consistent with a theory that paternally imprinted genes code for proteins involved in body size regulation, GRF1 knockout mice are smaller than their normal counterparts.27 Interestingly, suppression of the maternal allele in the brain is relaxed after approximately 3 weeks of age, concomitant with a dramatic increase in the expression of GRF1 during postnatal development of the brain.28,29 Alternative Forms of GRF Proteins

The mRNAs from approximately 80 genes are alternatively spliced, and GRF mRNAs are no exception. Although p140-GRF1 and p135-GRF2 are the major products of these genes in neurons, smaller mRNA transcripts have been detected by Northern blotting.29,30 Smaller protein isoforms have also been detected by Western blots for both proteins in a variety of tissues.6 However, in the brain, only a few alternative transcripts and associated protein isoforms have been confirmed. Figure 2A shows the organization of the GRF1 gene. It contains 28 exons that

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encode full-length p140-GRF1, which is expressed heavily in the adult brain. This is referred to as isoform 1 (Fig. 2A and 2B). A transcript (X59868) encoding isoform 2 was also found in the adult brain associated with a 2-kb mRNA transcript. A similar mRNA was also found in the adult human brain (Fig. 2A and 2B).30 This form encodes only the C-terminal 55 kD of the protein with its Ras-activating CDC25 domain, a neuronal domain that binds to NMDA-type glutamate receptors, and Ras exchange motif (REM) that stabilizes the CDC25 domain. Although detectable in the adult,16 Western blots suggest its level may be higher in younger animals.31 Another alternative smaller transcript (AK1403) (Fig. 2A) encoding isoform 3 was also found in the brain. This protein contains only a PH domain that is different from the one found in the N-terminus of isoform 1 because it includes a different exon 2, resulting in a different C-terminal half of the domain (Fig. 2B). A protein of similar content has been detected in the brain and pancreas, where it may function as a dominantnegative protein that blocks normal p140-GRF1 function.32 As described later, these smaller isoforms have different functional domains than p140GRF1; they clearly play different, but not understood, roles in brain function. Based on postings in databases, GRF2 may also have one alternative splice variant (Fig. 2C). It would encode the DH domain through part of the CDC25 domain (Fig. 2D). However, it has not been confirmed experimentally, and it was only detected in the thymus. Thus, it is not likely to be relevant to GRF2 function in the brain. Smaller forms of GRF2 have been detected at the protein level in tissues other than the brain, but they have not been assigned to specific mRNA transcripts or specific functions.7 Regulation of GRF Gene Expression

Both p140-GRF129 and p130-GRF2 (Feig, unpublished observations) are expressed in neurons, but not glia, in the brain. Both are expressed in all brain

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addition, cultures of newborn striatal neurons from GRF1 knockout mice display decreased dopamine and glutamate receptor signaling that may contribute to altered behaviors detected in adult animals.10 Finally, suppression of GRF1 expression causes defective associative olfactory memory in neonatal mice.35 Acute changes in p140-GRF1 and p130-GRF2 levels have been detected in response to a variety of neurological perturbations including psychostimulants and neurodegenerative stimuli, suggesting that alterations in Ras and Rac signaling downstream of these proteins participate in some neurological disorders. For example, cocaine, amphetamine, and Δ-9-tetrahydrocannibinol have all been shown to increase GRF1 expression in the striatum, cortex, and cerebellum,36-38 whereas overexpression of Alzheimer-related amyloid precursor protein suppresses GRF1 expression.39 Regulation of GRF Protein Stability

Figure 2. Gene organization of GRF1 and GRF2 including mRNA splice variants. (A) Exons of the GRF1 gene along with splice variants that lead to 3 different GRF1 protein isoforms. (B) Functional domains found in p140-GRF1 (isoform 1), p55-GRF1 (isoform 2), and p20-GRF1 (isoform 3). (C) Exons of the GRF2 gene and splice variants that potentially lead to 2 different GRF2 proteins. (D) Functional domains found in p135-GRF2 and p60-GRF2 (not definitively identified).

regions, although differences in intensity have been reported.33 A common feature of both p140-GRF1 and p130GRF2 is that their expression in the brain is developmentally regulated, such that very low levels are present in the neonate.28,29 Expression rises substantially from postnatal day 10 to day 30 and remains high throughout adulthood. Importantly, phenotypes of GRF

knockout mice are also age dependent. For example, the low levels of GRF expression found in young wild-type animals are not sufficient to support a role for GRF proteins in the regulation of synaptic plasticity in the hippocampus.34 In contrast, GRF1 knockout mice display reduced body size soon after birth because of an early defect in growth hormone production.27 In

GRF1 and GRF2 may be regulated at the level of protein stability in cells. Both proteins contain PEST sequences as well as cyclin destruction boxes between the CDC25 and REM domains (Fig. 1). In addition, p140-GRF1 can be phosphorylated by CDK5. This appears to promote its degradation in vivo, as CDK5 knockout mice display enhanced p140-GRF1 levels.40 How these events fit into its roles in specific brain functions remains to be determined.

Structural Organization and Biochemical Functions This discussion will focus only on p140GRF1 and p130-GRF2 because little is known about the function of the smaller isoforms of GRF1 or GRF2. Both p140GRF1 and p140-GRF2 transmit signals from multiple neurotransmitter receptors to downstream signaling cascades mediated by both Rac and Ras (and closely related M-Ras and TC21) proteins.41 To carry out these complex functions, p140-GRF1 and p135-GRF2 are large proteins containing 2 catalytic

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domains and multiple regulatory domains (Fig. 1). While p140-GRF1 and p130-GRF2 contain mostly the same domains, they have been found to function quite differently in vivo, a subject that will be addressed briefly below. An important regulatory domain on both proteins is the calcium-dependent, calmodulin-binding IQ motif that is involved in p140-GRF18 and p130GRF27 activation. Calmodulin binding to GRFs is calcium dependent,8 so that both proteins can activate Ras and Rac GTPases20,42 in response to increased calcium levels generated by specific neurotransmitter receptors.9,28 How calcium/ calmodulin binding increases the GEF activities of GRF proteins is not well understood. Some evidence suggests that the GRF N-terminus negatively regulates the Ras-GEF domain at its Cterminus, consistent with how the related Sos GEFs are regulated.43 However, structural studies have shown that whereas the SOS CDC25 domain is inhibited by upstream sequences, the CDC25 domain of GRF1 is constitutively in the active conformation.44 Moreover, the presence of bound calmodulin does not increase the intrinsic GEF activity of the protein as assayed in vitro.8 Some data suggest that calcium activates GRF proteins by inducing its movement from the cytoplasm to the membrane.20 However, a constitutively membrane-bound form of GRF1 does not have enhanced GEF activity in vivo compared to wild-type protein.45 Thus, activation of GRF proteins by calcium/ calmodulin binding most likely involves more subtle changes that bring them in close contact with Ras proteins. How this process is occurring in the context of neuronal cells remains to be identified. Just upstream of the IQ motif on both proteins is a coiled-coil motif and then the N-terminal PH domain. βγ subunits of G proteins can bind to the PH domain of GRF1, which may contribute to G protein–coupled receptor signaling through p140-GRF1.46 The amino acid sequence of the PH domain of GRF2 is

quite different from GRF1 (58% identity), and whether βγ can also bind to it has not been investigated. In addition, the PH, coiled-coil, and IQ motifs appear to function as a unit in localizing GRF1 to the membrane fraction of cells and allow it to respond to calcium signals.47 It also binds a set of scaffold proteins that targets the exchange factor to specific Rac effector proteins.48,49 Downstream, a DH/PH domain responsible for activating the Rac GTPase is also present on both proteins. This is followed by a REM domain that helps regulate the activity of the CDC25 domain in most Ras family GEFs. GRF1, but not GRF2, also has a “neuronal domain” that binds to NMDAtype glutamate receptors that contain NR2B subunits.50 Both proteins also contain cyclin destruction boxes and PEST sequences in the region between the REM- and Ras-activating CDC25 domains.7,51 Interestingly, the CDC25 domain of GRF1, but not GRF2, is also capable of activating the Ras-related R-Ras protein that is known to regulate inside-out integrin signaling,45,52 but its role in GRF1 function in neurons is not yet known. GRFs are known to be expressed in postsynaptic densities associated with the plasma membrane of the synapse, where it mediates the action of NMDA-type glutamate receptors.53 A significant amount of GRF proteins are also found in the cell soma of neurons, some of which are associated with the endoplasmic reticulum.54 At this membrane site, GRFs may be activated by signals distinct from those occurring in the plasma membrane, such as those from G protein receptors. GRF1 has 3 isoforms generated by alternative splicing of the GRF1 mRNA. Importantly, these isoforms likely play very different functions from full-length p140-GRF1. For example, because isoform 2 (p55-GRF1) has a functional Ras-activating domain but no IQ motif, it likely activates Ras in response to signals other than calcium. Moreover, it does not contain a Rac-activating DH

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domain, so that it activates a smaller set of effector signaling pathways than p140-GRF1. In fact, p135-GRF2 is a lot closer in domain makeup to p140-GRF1 than these GRF1 isoforms. Therefore, taking into account which isoforms of GRF1 are inhibited in knockout mice used to study GRF function is an important issue.

Transgenic Mice as Tools to Reveal GRF Functions Knockout transgenic mice have been used extensively to reveal the function of both GRF1 and GRF2 proteins. They have been the source of isolated neurons and acute brain slices that have revealed how GRF proteins participate in specific biochemical pathways, distinct forms of synaptic plasticity, and other neuronal cell processes. These mice have also been used to study the role of GRF proteins in specific animal behaviors as well as responses to psychoactive drugs and neurodegenerating stresses. However, as described below, significant differences in some phenotypes have been observed among various GRF1 knockout models generated (Fig. 3). While the reason has not been directly addressed, an important difference among some of these animal models is that they were generated by targeting different parts of the GRF1 gene (Fig. 3). As a consequence, they are predicted to block expression of different subsets of GRF isoforms. In particular, all 6 of the transgenic GRF1 models block expression of the fulllength p140-GRF1 protein (isoform 1). In contrast, all but 2 models are predicted to also block expression of at least one alternatively spliced form of its mRNA. For example, to make GRF1 knockout mice, Brambilla et al. (GRF1Brambilla),55 Itier (GRF1Itier),27 and Font de Mora et al. (GRF1Font de Mora)56 targeted the CDC25 domain of the GRF1 gene, which should block expression of isoform 2 along with isoform 1. Yoon et al. (GRF1Yoon)26 targeted the

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Another caveat associated with all of these studies is that they suppress GRF expression in all tissues. Thus, some of the phenotypes, particularly behavioral ones, could be due to the loss of GRF proteins in more than one region of the brain. For example, GRF1 knockout mice are small because of decreased growth hormone secretion from the pituitary,27 which raises the possibility that neuroendocrine alterations contribute to some of the observed phenotypes. Also, GRF1Font de Mora knockout mice display a significant defect in visual function beginning at 4 months of age, which could impact behavior studies in some GRF1 knockout models.60 Hopefully, future studies will conditionally suppress individual GRF isoforms in specific cell types to advance our understanding of these important proteins.

Receptors in Neurons that Function through GRF Proteins NMDA-Type Glutamate Receptors (NMDARs)

Figure 3. Properties of transgenic mice used in studies to reveal the function of GRF proteins.

promoter, which likely suppresses all isoforms. One GRF1 overexpression mouse (GRF1-OYoon)26 that reactivates the promoter of the normally inactive maternal allele should increase the expression of all GRF1 isoforms. Thus, it is not clear whether the phenotypes observed in these mice are from the loss/ overexpression of the well-characterized p140-GRF1 protein or the poorly characterized p55-GRF1 and p20-GRF1 proteins or any combination of them. In contrast, the mice generated by Giese et al. (GRF1Giese)57 targeted the DH domain, which should leave both isoforms 2 and 3 intact. In fact, RNA sequencing has confirmed that the mRNA for isoforms 2 and 3 are still expressed in

the adult hippocampus of these mice (Saavedra et al., unpublished observations). ENU-treated mice made by Clapcott et al. (GRF1Clapcott)58 have a mutation in the GRF1 gene that should also allow mRNA expression of isoforms 2 and 3. Thus, the phenotypes of these mice represent the loss of only p140-GRF1. Two knockout models for GRF2 have been developed. Tian et al. (GRF2Tian)28 inactivated exon 1, while FernandezMedarde et al. (GRF2Fernandez)59 inactivated exon 24. In both cases, the full-length p135-GRF2 protein is no longer expressed, but a putative smaller form (Fig. 3) is predicted to remain. However, the only evidence for its expression was in immune cells.

NMDARs are calcium-permeable receptors present at excitatory synapses, where glutamate is the neurotransmitter. They have been studied extensively in the hippocampus, where they have been shown to play critical roles in synaptic plasticity, learning, and memory.61 The discovery that GRF proteins mediate part of the functions of NMDARs came from 2 approaches. In one, a yeast 2-hybrid screen for proteins that bind to the NR2B subunit of NMDARs detected the C-terminal portion of GRF1 (termed the neuronal domain) (Fig. 1).50 Its functional significance was suggested by the finding that expression of the NR2B-binding domain of GRF1 in primary cultures of hippocampal neurons blocked NMDA activation of Erk MAP kinase, as detected by immunofluorescence quantification of p-Erk. Another approach took advantage of GRF1Giese knockout mice, where only the p140-GRF1 isoform is missing. This study used acute cortical slice

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Figure 4. p140-GRF1 and p135-GRF2 mediate opposite forms of synaptic plasticity in the CA1 region of the hippocampus of 1-month-old mice. p135-GRF2 mediates the ability of NR2Acontaining NMDARs to promote LTP at least in part through its ability to preferentially activate Ras and Erk MAP kinase. In contrast, p140-GRF1 mediates the ability of NR2B receptors to promote LTD at least in part through its ability to preferentially activate Rac and p38 MAP kinase. Some of this signaling specificity difference between GRF family members may derive from the local association of regulators of signaling downstream of GRF proteins, such as the negative regulator of Ras, Syn-GAP, which is known to associate with NR2B-containing NMDARs.

cultures to show partial suppression of NMDA-induced activation of Ras and Erk, as detected by active Ras pull-down assays and by immunoblotting for p-Erk, respectively. These studies also used GRF2Tian knockout mice in a similar manner to show that p135-GRF2 has an even larger role in coupling NMDARs to Ras and Erk in the cortex.28 The latter study also demonstrated a strong age dependence for GRF function in NMDAR signaling. Thus, slices from GRF1Giese and GRF2Tian knockout mice displayed defective NMDA activation of Ras and Erk only in mice over the age of approximately 25 days. This pattern is consistent with the known age dependence of GRF expression, where p140GRF1 and p135-GRF2 are expressed at low levels in neonatal animals and increase with age until weaning, after which they remain at high levels throughout adulthood.29,34 A similar agedependent effect on synaptic plasticity in the hippocampus was also found.34

Both p140-GRF1 and p135-GRF2 have the potential to also activate the Rac GTPase and its effector proteins through their DH domains. Nevertheless, while GRF1Giese knockout mice displayed defective NMDAR activation of the Rac effector, p38, in hippocampal slices from 1-month-old animals, GRF2Tian knockout mice did not.34 Overall, these data indicated that although both p140-GRF1 and p130GRF2 have the potential to mediate regulation of both Erk and p38 MAP kinases, in the context of the hippocampus, GRF2 plays a more dominant role in NMDAR activation of Erk, whereas GRF1 plays a more dominant role in NMDA activation of p38 MAP kinase (Fig. 4). As described previously, these roles are age dependent, such that no defect in Erk or p38 activation was observed in mice younger than approximately 25 days of age. The physiological significance of these differences is discussed below.

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Although the mechanism for this difference in signaling specificity has yet to be revealed, one hint has come from the observation that they mediate the activity of different subsets of NMDARs. NMDARs are heterotetramers containing dimers of both NR1 subunits that permeate calcium and dimers of regulatory NR2A to NR2D subunits. The predominant NR2 subunits comprising NMDARs in the mature hippocampus used in these studies are NR2A and NR2B. As mentioned previously, GRF1 has been shown to bind directly to NR2B but not NR2A subunits of NMDARs.50 Consistent with the idea that this preferentially couples p140-GRF1 to NR2Bcontaining receptors is the finding that NMDA activation of p38, but not Erk, is blocked by the treatment of hippocampal brain slices with a specific inhibitor of NR2B subunits, ifenprodil. Moreover, treatment with APV, which shows some selectivity for NR2A subunits, blocked NMDA activation of Erk but not p38.34 These findings suggest that downstream signaling specificity differences displayed by p140-GRF1 and p130-GRF2 are due, at least in part, to their functioning in different signaling complexes (Fig. 4). In support of this idea is the observation that Syn-GAP, a negative regulator of Ras, is associated specifically with NR2B-containing NMDA receptors,62 which could account for the suppressed Erk activation by p140-GRF1. Moreover, a set of scaffold proteins have been found to associate with p140-GRF1, which target it to specific Rac effectors.47,48,63 One such scaffold, JIP2, directs GRF1 to the Rac effector p38. If it is found to associate with GRF1, but not GRF2, the potency of the former but not the latter GEF to activate p38 could be explained. By analogy, it will be worthwhile investigating whether a Rac-GAP and a scaffold for Erk activation are associated specifically with GRF2 and NR2Acontaining NMDARs to favor Ras/Erk over Rac/p38 signaling. Such a model opens the possibility that GRF proteins may be used differently in other

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synapses that might express different scaffolds and accessory proteins. The consequence of this difference in downstream signaling specificity on the roles of GRF1 and GRF2 in synaptic plasticity, learning, and memory is discussed below. Calcium-Permeable, AMPA-Type Glutamate Receptors (CP-AMPARs)

AMPA-type glutamate receptors (AMPARs) are heterotetromeric complexes composed of GluA1 to GluA4 subunits. They are best known for their ability to permeate sodium into the dendrites of the postsynaptic cell after binding glutamate released from the presynaptic axon. This contributes to the depolarization of the postsynaptic cell as part of synaptic transmission. However, a subclass of AMPARs that do not contain GluA2 subunits also permeate calcium and thus can contribute to many postsynaptic signaling pathways.64,65 CP-AMPARs were first shown to function in inhibitory interneurons.66 More recently, however, they have been shown to contribute to synaptic plasticity in excitatory neurons of the hippocampus. They often are induced to enter the synapse transiently and participate in the early stages of a classic and important type of synaptic plasticity, longterm potentiation (LTP), which is known to be a cellular phenomenon that contributes to learning and memory.67-69 However, this role may be stimulus and age dependent.70 Which downstream signaling pathways are regulated by CPAMPARs to carry out their functions is not understood. Studies on GRF1Giese and GRF2Tian knockout mice have demonstrated that both proteins have the potential to mediate some aspects of CP-AMPAR function.9 In particular, AMPA stimulation of cortical brain slices, which occurs through CP-AMPARs, was suppressed partially in either GRF1 or GRF2 knockout cortical brain slices and completely in double knockout samples. Moreover, both p140-GRF1 and p135-GRF2 were


immunoprecipitated with GluA1 (in receptors that can permeate calcium if GluA2 is not present) but not GluA2 subunits (not in receptor complexes that permeate calcium), only after AMPA stimulation of cortical brain slices. Similar to NMDAR signaling, CPAMPAR signaling through GRFs is age dependent, such that AMPA activation of Erk is not GRF dependent in young animals. Interestingly, coupling of CPAMPARs to GRF proteins only begins after postnatal day 30, 5 days after NMDARs begin to signal through GRFs. This could be related to the observation that CP-AMPARs do not contribute to LTP until mice are 8 weeks of age.69,71 How CP-AMPAR signaling through GRFs contributes to synaptic plasticity or specific behaviors in animals remains to be determined. D1/5 Dopamine Receptors

Dopamine receptors are a family of G protein–coupled receptors that are found throughout the CNS. The D1 and D5 subtypes of dopamine receptors function through G proteins, which activate adenylate cyclase to elevate cellular cAMP levels. D1/5 receptors have been studied extensively in the dorsal striatum, a region of the brain that plays an important role in responses to a variety of psychostimulants including cocaine. Studies on in vitro cultures of neurons from the striatum of neonatal GRF1Brambilla knockout mice, mice containing a mutageninduced nonsense mutation, GRF1Clapcott, as well as mice overexpressing GRF1 (GRF1-OYoon), have shown that GRF1 regulates the activation of Erk in response to agonists of D1/5 dopamine receptors (Fig. 5).10 The biochemical mechanism by which D1/5 receptors activate GRF1 has not been revealed, although it may be through PKA-induced phosphorylation of GRF1 (Fig. 5). Muscarinic-Type Acetylcholine Receptors

Early studies on GRF1 using NIH3T3 and COS-7 cells showed that

transfection of subtype 1 muscarinic receptors activated cotransfected p140GRF1’s (which is not normally expressed in these cells) ability to activate Erk upon charbachol addition.72 Whether this activation mechanism was due to the observed phosphorylation of GRF1 (see below) or elevated calcium is not clear. Also, whether this signaling cascade is functional in CNS neurons has yet to be established. TrkB Receptors

A yeast 2-hybrid screen revealed that the Trk receptors, TrkA, TrkB, and TrkC, can bind to the N-terminal PH domain of p140-GRF1. Upon binding, these receptors phosphorylate GRF1 on unspecified tyrosine(s) in its N-terminal PH domain. Moreover, transfection of GRF1 synergizes with TrkBA to promote PC12 differentiation into neurons, as assessed by neurite outgrowth. This occurred in a manner dependent upon the 2 proteins binding to each other, suggesting that they may work in concert to alter neuronal function.73 However, it should be kept in mind that GRF1 is not normally expressed in PC12 cells, and all GRF1 knockout mice studied show no significant defect in neuronal development. Thus, if these 2 proteins function together in neurons, it must be for a function other than to promote differentiation. Moreover, it remains to be determined whether these 2 proteins interact in CNS neurons, where they are naturally expressed, and whether GRF1 does, in fact, get tyrosine phosphorylated by Trk receptors in CNS neurons in vivo.

Regulation of GRF Function by Phosphorylation GRF proteins have been shown to be phosphorylated at specific sites by a variety of intracellular kinases. In some cases, the biochemical consequence of this event has been described, but how phosphorylation contributes to a specific role in synaptic plasticity, behavior, or responses to drugs remains to be revealed.

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the DH domain of GRF1 prevented calcium activation of Erk by GRF1 transfected into COS cells.78 How these phosphorylation events contribute to GRF1 and GRF2 function in the brain must await the analysis of the biological activity in vivo of CDK5 phosphorylation-resistant mutant GRFs. PKA and CamKI

Figure 5. p140-GRF1 integrates glutamate and dopamine signaling in the striatum. GRF1 mediates Ras/Erk activation by both glutamate and dopamine receptors. The former is thought to function through calcium (Ca2+) entering through NR2B-containing NMDARs and the calmodulin (CaM)–binding domain of p140-GRF1 and/or p55-GRF1 (not shown). Dopamine is thought to function through cAMP/PKA to phosphorylate GRF1. The role of the DH domain in this process has not been investigated.

CDK5

Cyclin-dependent kinase 5 (CDK5) was originally thought to be a member of a family of kinases that is regulated by binding cyclin proteins. Instead, CDK5 is activated by binding either of 2 proteins, p35 and p39. CDK5 is expressed predominantly in the brain and has been implicated in a plethora of neuronal functions. It is critical for proper neuronal development, neurite outgrowth, and neuronal migration during development and axon patterning. It participates in synaptic vesicle release through its interaction with synapsin and Munc-18. Relevant to GRF function is the fact that CDK5 is also involved in dopamine activation of Erk through the phosphorylation of DARPP-32.74 Like GRF1, it is involved in adaptive changes associated with cocaine75 and synaptic plasticity associated with learning and memory.76 Thus, it is of interest that CDK5 has been shown to be capable of phosphorylating both p140-GRF1 and p135-GRF2 by virtue of the fact that p35 binds to these GEFs delivering CDK5 to this

substrate.40,77 GRF1 stability has been found to be reduced upon phosphorylation by CDK5 on Ser 731, which resides between the CDC25 and REM domains (Fig. 1). In fact, CDK5 knockout mice express higher than normal levels of GRF1. Elevated GRF1 levels in cultured neurons lead to condensed nuclei. How this relates to GRF1 function in neurons remains to be determined.77 CDK5 also phosphorylates GRF2 at the same site.40 However, this does not affect the protein’s stability. Rather, it alters the way this protein signals downstream, at least in transfected tissue culture cells, where p135-GRF2 is not normally expressed. CDK5-induced phosphorylation of p135-GRF2 leads to decreased activation of Rac via its DH domain, but not Ras via its CDC25. However, Erk activation is suppressed, indicative of the permissive role Rac plays in Erk activation in this system. Supporting the idea that regulation of the Rac-activating DH domain of GRF proteins can influence Erk activation is a previous finding that a point mutation in

p140-GRF1 is known to be phosphorylated at position S916 after cellular stimulation by a variety of ligands (Fig. 5). It was originally detected after transfection of tissue culture cells with type 1 muscarinic receptors along with GRF1.72 This was associated with a small but significant increase in the intrinsic GEF activity against Ras. Other stimuli in tissue culture that lead to this phosphorylation event include lysophosphatidic acid.79 Moreover, phospho-specific antibodies to this site in GRF1 demonstrated that this site does get phosphorylated in the dendrites of cortical neurons in the brain, implying that this phenomenon plays some role in synaptic plasticity.80 Interestingly, this site on GRF1 was shown to become phosphorylated in response to signals that induce LTP in the CA1 region of the hippocampus.81 This event was shown to involve CamKI, although PKA was likely the enzyme that directly phosphorylates GRF1. Inhibition of GRF1 phosphorylation by inhibitors to CamKI suppressed LTPinduced Erk activation, suggesting that this event contributes to LTP induction. However, studies of GRF1Giese knockout mice found that GRF1 is not needed for LTP induction in this region of the hippocampus.34 A clearer understanding of the role of this phosphorylation event on GRF1 will have to wait for experiments that test the biological activity of a S916 phospho-resistant mutant in vivo. Tyrosine Phosphorylation

Trk receptors can phosphorylate GRF1, but whether they do so in vivo remains to be determined. Other tyrosine kinases including Src and Ack1 can

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phosphorylate GRF1 and change its activity.82,83 In particular, Src has been reported to increase GRF1 activity on Rac presumably through its DH domain, while Lck and Ack have been shown to increase its activity on Ras. However, the specific sites of phosphorylation have not been revealed, and whether these events occur in the brain is also not known.

Region-Specific Functions of GRF Proteins in the Central Nervous System Neither GRF1 nor GRF2 knockout mice show gross alterations in brain morphology, implying that they do not play an important role in neurodevelopment.5,59 This is consistent with their expression being developmentally regulated with low levels of expression, appearing first after birth and increasing to maximal levels in the adult.28,29 However, both p140-GRF1 and p135-GRF2 protect brain structure, as GRF1Giese/GRF2Tian knockout mice display enhanced damage in response to stroke-induced ischemia.28 This phenomenon requires deletion of both GRF genes, implying that GRF1 and GRF2 play cooperating functions in this setting possibly via activation of the CREB transcription factor. In addition, GRF1Brambilla knockout mice are more susceptible to druginduced seizures most likely because hippocampal neurons display enhanced basal intrinsic excitability.84 However, this phenotype was not detected in GRF1Giese knockout mice, which lack only p140-GRF1, while GRF1Brambilla mice are predicted to also lack p55-GRF1. Thus, it is possible that the enhanced hippocampal excitability observed in the GRF1Brambilla knockout mice is due to the loss of p55-GRF1, not p140-GRF1. How this small form of GRF1 might normally suppress neuron excitability remains to be determined. Studies on GRF knockout mice have also found distinct functions for these proteins in different regions of the brain including


the pituitary, hippocampus, amygdala, striatum, olfactory bulb, cerebellum, and retina.

both p140-GRF1 and p55-GRF1, while GRF1Giese mice lack only the former, it is possible that the contribution to amygdala memory is from p55-GRF1.

Pituitary

The earliest phenotype observed for GRF1 knockout mice was soon after birth, when it becomes apparent that they have a smaller than normal body size. This phenotype is likely due to reduced levels of IGF-185 and its pituitary-derived regulator, growth hormone.27 This phenotype was reported for GRF1Itier,27 GRF1Font de Mora,56 GRF1Clapcott,58 and GRF1Giese.57 For reasons that are not clear, this phenotype was not reported for GRF1Brambilla 55 and GRF1Yoon.25 How GRF1 regulates this process remains unknown. GRF2 knockout mice do not show alterations in body size, but whether GRF2 plays a more subtle role in neuroendocrine function cannot be ruled out. Amygdala

Synaptic plasticity. In complex vertebrates, including humans, the amygdala, which is part of the basal ganglia, functions in the formation and storage of memories associated with emotional events. GRF1Brambilla knockout mice show a defect in both synaptic plasticity and behavior associated with the function of the amygdala. In particular, defective theta burst–induced LTP was observed in the basolateral amygdala.55 Which downstream effectors of GRF1 mediate LTP in the amygdala remains to be determined. Behavior. GRF1Brambilla knockout mice show normal learning and short-term memory but are impaired in memory consolidation, particularly those associated with emotional conditioning tasks known to require a functional amygdala.55 However, GRF1Giese knockout mice showed no amygdala-dependent memory defects.57 The reason for this discrepancy is not clear. However, because GRF1Brambilla knockout mice lack

Hippocampus

The hippocampus is known to play an important role in new spatial memory formation. It develops relatively late, and its contribution to memory begins only at preadolescence (~1 month of age) in rodents.86 Because neurons of the hippocampus are organized in discreet layers that interact in a relatively simple organization, it has been used extensively as a model system to study how neuron function contributes to learning and memory. Within the hippocampus, information flow is largely unidirectional, with signals from the entorhinal cortex (EC) propagating through a series of tightly packed cell layers to the dentate gyrus, the CA3 layer, the CA1 layer, the subiculum, and finally out of the hippocampus back to the EC. Each of these regions of the hippocampus is thought to contribute differently to its overall function. Synaptic plasticity. GRF1Giese and GRF2 knockout mice have been used to show that p140-GRF1 and p135-GRF2 contribute to opposing forms of synaptic plasticity at the extensively investigated CA3-CA1 synapse of the hippocampus of 1-month-old mice.34 In particular, GRF2 knockout mice have a defect in LTP, a long-lasting increase in synaptic signaling efficiency that is known to play a role in memory formation, but no defect in LTD, a long-lasting decrease in synaptic signaling efficiency whose role in memory formation is less clearly defined. In contrast, GRF1 knockout mice have a defect in LTD but not in LTP. These phenotypes are due to the loss of p135GRF2 and p140-GRF1, respectively, and not to other GRF isoforms or indirect effects of the loss of GRFs in other regions of the brain. This is based on the finding that reconstitution of these proteins specifically in CA1 hippocampal Tian

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neurons of 1-month-old mice by stereotaxic injection of GRF-expressing viruses led to restored LTP and LTD, respectively (Jin and Feig, unpublished data). GRF1Brambilla knockout mice also show no defect in LTP in the CA1 hippocampus,55 but LTD assays were not performed. GRF1Brambilla mice did show an LTP defect in the amygdala. However, unlike LTP in the CA1 hippocampus, LTP in the amygdala is not NMDAR dependent. Instead, it is dependent upon muscarinic receptors.87,88 These findings highlight how GRF proteins may play different functions at synapses from different parts of the brain. How do GRF1 and GRF2 play opposing roles in synaptic plasticity in the hippocampus? One likely reason is the ability of GRF1 and GRF2 to couple NMDARs to different MAP kinase family members. p135-GRF2 predominantly mediates Erk MAP kinase activation, which is known to participate in LTP induction. In contrast, p130GRF1 predominantly mediates p38 MAP kinase activation, which is known to promote LTD (Fig. 4). Another significant difference between p140-GRF1 and p140-GRF2 is that they mediate the actions of different subtypes of NMDARs on the induction of synaptic plasticity in the CA1 hippocampus (Fig. 4). NMDARs consist of dimers of calcium-permeable NR1 subunits bound to either homodimers or heterodimers of regulatory NR2 subunits, NR2A to NR2D.89 The rationale for the existence of distinct subsets of receptors containing different NR2 subunits has been the subject of much controversy and remains poorly understood. In particular, some studies claimed that NR2A-containing receptors are specific for LTP induction, while NR2B receptors are specific for LTD induction.90,91 However, other studies have contradicted these findings.92-95 A general consensus has emerged that either subtype of receptor can induce LTP, with the key determinant being the magnitude of calcium influx through either channel, with LTP requiring more calcium than LTD.

One undisputed distinction between NMDAR subtypes is their calcium channel–gating properties. NR2B-containing receptors are slower to deactivate and therefore may carry more calcium per unit current than NR2A receptors (see review96). This has led to the hypothesis that NR2B receptors induce LTP more easily than NR2A receptors, and thus, the NR2A/NR2B ratio may control LTP/LTD thresholds. In fact, the ratio of these receptors is known to change with development and experience.97-99 Studies on GRF1Giese and GRF2Tian knockout mice demonstrate that NR2Acontaining receptors induce LTP differently than NR2B receptors. NR2A receptors induce LTP through p135GRF2,34,100 while NR2B-containing receptors do it through neither GRF1 nor GRF2. These studies also suggest that NR2B-containing receptors induce LTD through p140-GRF1, while NR2A-containing receptors mediate LTD in a GRF-independent mechanism. These observations suggest a model in which the NR2A/NR2B ratio in synapses not only determines the ease at which LTP can be induced,101 but it also generates qualitative differences in how LTP is generated via subunit-specific coupling to distinct intracellular signal transduction pathways. The physiological consequence of producing LTP or LTD by different biochemical pathways remains to be revealed. Interestingly, GRF1 and GRF2 begin to contribute to synaptic plasticity at this synapse only after mice are approximately 1 month of age,34 concomitant with the onset of hippocampal-dependent learning and memory. Before this time, the tyrosine kinase–responsive Sos exchange factors appear to be involved in coupling NMDARs to MAP kinases.28 This pattern implies that SOS exchange factors are involved in synaptic plasticity that is used for hippocampus development, while GRF exchange factors contribute to synaptic plasticity that is used for spatial learning and memory. Both Sos and GRF proteins have the

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capacity to activate Ras and Rac, so that how they contribute to different aspects of hippocampus function also remains to be determined. Behavior. A very well-established learning and memory paradigm that is dependent upon the hippocampus is contextual fear conditioning. In this assay, mice are placed in a specific context (a box with unique spatial cues) and then exposed briefly to an electrical shock to their feet. When mice are placed back in this context, they anticipate being shocked and freeze their movement. When this assay was performed on GRF1Giese knockout mice, they behaved like wild-type mice. But when mice were exposed to a more challenging test, the ability to distinguish between 2 closely related contexts, one in which they were shocked and one in which they were not, they failed.57 This process called “contextual discrimination” is known to be very dependent upon the hippocampus but not the amygdala.102 These mice were also defective in 2 other hippocampal-dependent behaviors, social transmission of food preference and the Morris water maze. The former test is based on the fact that mice develop a preference for foods that they recently smelled on the breath of other mice (“demonstrator” mice). Performance immediately after the interaction with the “demonstrator” mice is not sensitive to hippocampal lesions. However, 24 hours after the interaction with the “demonstrator” mice, performance on this task is hippocampus dependent.103 In this test, the GRF1Giese mice lose their preference for specific foods faster than wild-type mice. In the hidden-platform water maze, GRF1Giese mice needed more time than control littermates to reach the platform. Whether these behavioral defects are due to decreased hippocampal LTD in the CA1 or some other defect in hippocampal function remains to be determined. For reasons that are not clear, GRF1Brambilla knockout mice performed normally in this water

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maze test.55,103 GRF1Brambilla mice were not tested for contextual discrimination. Behavioral assays for GRF2 knockout mice have been tested less extensively than GRF1 mice. In fact, published reports have only involved double GRF1Giese/GRF2Tian mice, which showed a defect in standard contextual fear conditioning.104 Because GRF1Giese mice display normal contextual fear conditioning (only defective contextual discrimination), this phenotype is likely due to the loss of p135-GRF2 in these mice. In fact, single GRF2Tian knockout mice do display defective contextual fear conditioning (Jin and Feig, unpublished data). Finally, a growing body of data shows that the environment in which animals live can have a profound effect on the biochemistry of signaling pathways in the brain and alter behavior. For example, exposure of early adolescent mice, but not adult mice, to an enriched environment that includes more social interaction, novel objects, and voluntary exercise enables an otherwise latent NMDAR/p38 MAP kinase pathway that promotes LTP in the CA1 hippocampus. The presence of this signaling pathway restores LTP (but not LTD) to normal levels in the hippocampus of GRF1Giese/ GRF2Tian knockout mice. It also restores contextual fear memory to that found in normal mice.105 Striatum

Ras-GRF1 function has been studied in the striatum, part of the basal ganglion that resides in the inside part of the forebrain. It plays an important role in a variety of cognitive processes, including planning and modulation of movement, pathways that when altered can contribute to Parkinson and Huntington diseases. It is also involved in habit formation and drug addiction (see review106). Biochemical analysis of primary cultures of dissociated striatal neurons or organotypic cultures from neonatal GRF1Brambilla and GRF1Clappcott knockout mice revealed defective activation of Erk induced by either glutamate or agonists of D1/5 dopamine receptors.10 Interestingly,


inhibitors of NMDA receptors blocked not only glutamate-induced Erk activation but also dopamine-induced Erk activation. An analogous phenomenon was observed when a dopamine receptor antagonist was used after glutamate stimulation. This suggests crosstalk between receptor systems and that p140-GRF1 participates in the integration of 2 important signaling pathways in immature neurons. Cocaine is known to stimulate Erk in a variety of brain structures, including the striatum, nucleus accumbens, amygdala, and cingulated cortex, each of which is thought to contribute to its psychostimulatory effects.107 GRF1Brambilla and GRF1Clappcott knockout mice showed a decreased Erk response to cocaine in the dorsal striatum. GRF1-OYoon mice, which overexpress GRF1 proteins, showed an increased Erk response.10 Consistent with these biochemical results, long-term (but not short-term) effects of cocaine treatment on locomotion were influenced in a similar way; GRF1Brambilla mice showed decreased locomotion, and GRF1-OYoon mice showed increased locomotion. Parkinson disease is caused by the loss of dopaminergic neurons in the substantia nigra, which causes decreased dopamine signaling in the striatum. Dopamine replacement therapy leads to L-dopa–induced dyskinesia (LIDA), a disabling side effect. Strikingly, GRF1Brambilla knockout mice show a decrease in motor symptoms associated with LIDA presumably because of its role in mediating dopamine signaling to Erk in these cells.108 Because all of the methods used in this study to block GRF1 expression would be expected to suppress both p140-GRF1 and p55-GRF1 isoforms, which one(s) contribute to this phenotype remains to be determined. Olfactory Bulb

GRF1 is a paternally imprinted gene until weaning. As part of an effort to investigate the consequence of altering imprinting, a GRF1-null mouse, GRF1Yoon, was generated. A defect in an associative olfactory task, which is

known to be dependent only on the olfactory bulb, was found in neonatal mice.35 How GRF1 contributes to this process remains to be determined. In addition, because these mice are predicted to lack p140-GRF1, p55-GRF1, and p20-GRF1, it is not clear which GRF1 isoform is responsible for this function in neonatal neurons. Cerebellum

GRF1Brambilla knockout mice have been shown to display decreased desensitization to hypolocomotion induced by the administration of Δ-9-tetrahydrocannabinoid (THC).109 This was associated with a block in the natural downregulation of the THC receptor that takes place in the cerebellum. These effects were also seen with pharmacological inhibition of Erk. Thus, either p140-GRF1 or p55-GRF1 likely mediates THC receptor levels via regulation of Erk in the cerebellum. Retina

Studies on GRF1Font de Mora knockout mice have revealed an age-dependent defect in the retina. Beginning at 4 months of age, significant reductions in the wave amplitude of electroretinagrams of knockout mice were found compared to control mice, implying decreased light perception as mice age.60 Which missing signaling pathways mediated by GRF1 contribute to this defect was not revealed. However, microarray comparison of retina mRNA from wild-type and knockout mice showed differences in the expression of approximately 44 genes, some of which could be implicated in altered neuronal function. In addition, the expression of some of these genes showed the same age-dependent changes (i.e., first detected at 4 months of age) as changes in retina function. Because GRF1Font de Mora knockout mice are predicted to lack both p140-GRF1 and p55-GRF1, it will be important to determine which of these GRF1 isoforms is involved in this visual phenotype because visual alterations could impact behavior assays on

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mature mice in some of the mouse models studied to date. Interestingly, the GRF1 gene is in a locus of the genome that has been implicated in predisposition to myopia, raising the possibility that alteration in the expression of one or more of the 3 GRF1 isoforms has a role in a common human eye disorder.110

Conclusions Studies to date have shown that RasGRF proteins are important components of signaling pathways that mediate a variety of CNS functions, including hormonal production controlling body size, regulation of synaptic plasticity, and generation of specific forms of learning and memory. GRF proteins also have been found to contribute to neuronal survival in response to ischemia and participate in specific responses to psychoactive drugs. Future studies using more sophisticated techniques that allow individual GRF isoforms to be studied in a cell type–specific manner in vivo will undoubtedly enhance our understanding of the functions already obtained and reveal new roles for these multifunctional signaling molecules. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding This work was supported by the National Institutes of Health [grant number NIMH-R01MH083324].

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