A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

Chromatin modification is an important epigenetic mechanism underlying neuroplasticity. Histone methylation and acetylation have both been shown to modulate gene expression, but the machinery responsible for mediating these changes in neurons has remained elusive. Here we identify a chromatin-modifying complex containing the histone demethylase PHF8 and the acetyltransferase TIP60 as a key regulator of the activity-induced expression of Arc, an important mediator of synaptic plasticity. Clinically, mutations in PHF8 cause X-linked mental retardation while TIP60 has been implicated in the pathogenesis of Alzheimer's disease. Within minutes of increased synaptic activity, this dual function complex is rapidly recruited to the Arc promoter, where it specifically counteracts the transcriptionally repressive histone mark H3K9me2 to facilitate the formation of the transcriptionally permissive H3K9acS10P, thereby favoring transcriptional activation. Consequently, gain-of-function of the PHF8-TIP60 complex in primary rat hippocampal neurons has a positive effect on early activity-induced Arc gene expression, whereas interfering with the function of this complex abrogates it. A global proteomics screen revealed that the majority of common interactors of PHF8 and TIP60 were involved in mRNA processing, including PSF, an important molecule involved in neuronal gene regulation. Finally, we proceeded to show, using super-resolution microscopy, that PHF8 and TIP60 interact at the single molecule level with PSF, thereby situating this chromatin modifying complex at the crossroads of transcriptional activation. These findings point toward a mechanism by which an epigenetic pathway can regulate neuronal activity-dependent gene transcription, which has implications in the development of novel therapeutics for disorders of learning and memory.

Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation(1,2,3).

Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the ß-spectrin isoform ßSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and ßSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation.

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans.

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers.

Liver injury activates quiescent hepatic stellate cells (Q-HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF-HSC) sometimes causes cirrhosis and liver failure. However, MF-HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q-HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre-recombinase with ROSA-loxP-stop-loxP-green fluorescent protein (GFP) mice to generate GFAP-Cre/GFP double-transgenic mice. These mice were fed methionine choline-deficient, ethionine-supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP-expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q-HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP-expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Arc/Arg3.1 translation is controlled by convergent N-methyl-D-aspartate and Gs-coupled receptor signaling pathways.

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

Hepatic accumulation of Hedgehog-reactive progenitors increases with severity of fatty liver damage in mice.

Progenitors regenerate fatty livers but the mechanisms involved are uncertain. The Hedgehog pathway regulates mesendodermal progenitors and modulates mesenchymal-epithelial interactions during tissue remodeling. To determine if Hedgehog signaling increases in liver progenitors during fatty liver injury, we compared expression of Hedgehog ligands and target genes across a spectrum of injury. Leptin-deficient ob/ob mice with fatty livers and their healthy lean littermates were studied before and after exposure to the hepatotoxin, ethionine. At baseline, ob/ob mice had greater liver damage than controls. Ethionine induced liver injury in both ob/ob and lean mice, with greater injury occurring in ob/ob mice. After ethionine, the ob/ob mice developed liver atrophy and fibrosis. Liver injury increased hepatic accumulation of progenitors, including ductular cells that produced and responded to Hedgehog ligands. A dose-response relationship was demonstrated between liver injury and expansion of Hedgehog-responsive progenitors. In severely damaged, atrophic livers, nuclei in mature-appearing hepatocytes accumulated the Hedgehog-regulated mesenchymal transcription factor, Gli2 and lost expression of the liver epithelial transcription factor, hepatocyte nuclear factor 6 (HNF-6). Hepatic levels of collagen mRNA and pericellular collagen fibrils increased concomitantly. Hence, fatty liver injury increases Hedgehog activity in liver progenitors, and this might promote epithelial-mesenchymal transitions that result in liver fibrosis.

Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arc's molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the betaIV spectrin splice variant betaSpIVSigma5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to beta-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and betaSpIVSigma5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

The nonkinase phorbol ester receptor alpha 1-chimerin binds the NMDA receptor NR2A subunit and regulates dendritic spine density.

Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie synaptic plasticity. The key regulators of cytoskeletal reorganization in dendrites and spines are the Rho GTPases, which modify actin polymerization in response to synaptic signaling. Rho GTPase activity is modulated by multiple regulatory proteins, some of which have been found to associate with proteins localized to spines. Here, we show that the nonkinase phorbol ester receptor alpha1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. Alpha1-chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of alpha1-chimerin in cultured hippocampal neurons inhibits formation of new spines and removes existing spines. This reduction in spine density is mediated by Rac1 inhibition, because it depends critically on the presence of a functional GAP domain. Conversely, depletion of alpha1-chimerin leads to an increase in spine density, indicating that a basal inhibition of Rac1 maintains the number of spines at a submaximal level. The ability of alpha1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an alpha1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that alpha1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.

Translation of an integral membrane protein in distal dendrites of hippocampal neurons.

Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons.

Rapid dendritic transport of TGN38, a putative cargo receptor.

Protein transport to and from the postsynaptic plasma membrane is thought to be of central importance for synaptic plasticity. However, the molecular details of such processes are poorly understood. One mechanism by which membrane and secretory proteins may be transported to and from postsynaptic membranes is via cargo receptors. We studied the dendritic transport of TGN38, a putative cargo receptor thought to mediate protein transport between the trans-Golgi network (TGN), endosomes, and the plasma membrane. With fluorescence time-lapse imaging of neurons expressing a TGN38-green fluorescent protein fusion protein (GFP-TGN38), we observed rapid bidirectional dynamics of the protein in dendritic shafts. In addition, the protein was present on the surface and on intracellular membranes of dendrites and dendritic spines. Finally, GFP-TGN38 was found to cycle rapidly between the plasma membrane and intracellular membranes within dendrites, including those of spines. Together, our results suggest a role for TGN38 in facilitating rapid changes in the protein composition of postsynaptic membranes.

Ligand-binding residues integrate affinity and efficacy in the NMDA receptor.

The interaction of an agonist with its receptor can be characterized by two fundamental properties, affinity and efficacy. Affinity defines how tightly the agonist associates with its receptor, and efficacy measures the ability of the bound ligand to activate the receptor. Although affinity and efficacy are independent properties, the binding and activation processes that they describe are tightly coupled. This strong coupling has complicated the interpretation of concentration-response phenotypes caused by receptor mutations. We present an approach that quantifies the role of individual amino acids in defining affinity and efficacy. This method, which employs partial agonists and covalent modification of introduced cysteines, was applied to the ligand-binding sites of the NMDA receptor. Recent crystallographic structures for glutamate receptor ligand-binding cores allowed identification of residues that are either known or are predicted to be critical for ligand binding in the NR1 and NR2A subunit, respectively. Mutation of amino acids whose sidechains would directly coordinate bound ligands affected both agonist affinity and efficacy. In contrast, positions predicted to stabilize the closed-cleft conformation contributed only to agonist efficacy. The results provide a molecular basis for the tight coupling of agonist binding and receptor activation.

The identification of a second actin-binding region in spinophilin/neurabin II.

Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

Effects of mRNA untranslated regions on translational efficiency of NMDA receptor subunits.

Because NMDA receptors play critical roles in both neuronal survival and plasticity, their expression levels need to be carefully controlled. The number of functional NMDA receptors is temporally and spatially regulated at a hierarchy of levels, from gene transcription to protein trafficking. In this review we will focus on mechanisms for controlling functional expression of NMDA receptors that involve altering the efficacy of mRNA translation. One advantage of this level of control is that new receptors can be generated both rapidly and locally in response to appropriate synaptic activity patterns.

The NMDA receptor M3 segment is a conserved transduction element coupling ligand binding to channel opening.

Ion channels alternate stochastically between two functional states, open and closed. This gating behavior is controlled by membrane potential or by the binding of neurotransmitters in voltage- and ligand-gated channels, respectively. Although much progress has been made in defining the structure and function of the ligand-binding cores and the voltage sensors, how these domains couple to channel opening remains poorly understood. Here we show that the M3 transmembrane segments of the NMDA receptor allosterically interact with both the ligand-binding cores and the channel gate. It is proposed that M3 functions as a transduction element whose conformational change couples ligand binding with channel opening. Furthermore, amino acid homology between glutamate receptor M3 segments and the equivalent S6 or TM2 segments in K(+) channels suggests that ion channel activation and gating are both structurally and functionally conserved.