A neurotoxic dose of methamphetamine induces gene expression of Homer 1a, but not Homer 1b or 1c, in the striatum and nucleus accumbens

Homer proteins, which regulate the signaling pathway of metabotropic glutamate receptors, may contribute to the glutamatergic modulation of dopamine neurons in the basal ganglia. This study examined whether the induction of Homer 1 genes is or not associated with the methamphetamine-induced dopaminergic neurotoxicity in the discrete brain regions of rats. Basal levels of Homer 1a and 1c mRNAs in the forebrain regions were higher than those in the substantia nigra, whereas Homer 1b mRNA levels were higher in the substantia nigra than those in the forebrain regions examined. A neurotoxic dose (40 mg/kg, i.p.) of methamphetamine increased the mRNA and protein levels of Homer 1a in the striatum and nucleus accumbens, but not in the medial prefrontal cortex or the substantia nigra. Both Homer 1b and 1c mRNAs were not affected in any brain regions examined. These results suggest that the induction of Homer 1a gene may be involved at least in part in the methamphetamine-induced dopaminergic neurotoxicity, possibly through the glutamate-dopaminergic interaction.     

Rybp interacts with Hippi and enhances Hippi-mediated apoptosis

Rybp (DEDAF) has been shown to interact with DED-containing proteins and to encode pro-apoptotic functions. Here we characterize a novel interaction between Rybp and Hippi, a protein implicated in neuronal apoptosis as well as in the pathogenesis of Huntington’s disease. Rybp can synergize with Hippi to enhance Caspase 8-mediated apoptosis and also appears to be essential for Hippi-mediated apoptosis. Moreover, Rybp may mediate or regulate the interaction between Hippi and Caspase 8. Finally, Rybp and Hippi co-localize in a subset of neurons in the developing mouse brain. Together, these findings suggest that Rybp and Hippi may functionally interact in the apoptotic processes that accompany normal murine neural development.     

Protective effect of Homer 1a on tumor necrosis factor-α with cycloheximide-induced apoptosis is mediated by mitogen-activated protein kinase pathways

Although Homer 1, of the postsynaptic density, regulates apoptosis, the signaling mechanisms are not fully elucidated. In this study, we found that tumor necrosis factor-α (TNF-α)/cycloheximide (CHX) treatment transiently increased Homer 1a (the short variant of Homer 1), but did not affect Homer 1b/c (the long variant of Homer 1). Overexpression of Homer 1a blocked TNF-α/CHX-induced apoptotic cell death, whereas inhibition of Homer 1a induction enhanced the pro-apoptotic effect of TNF-α/CHX treatment. Moreover, brain-derived neurotrophic factor, as a potential activator of endogenous Homer 1a, inhibited apoptotic cell death after TNF-α/CHX treatment through induction of Homer 1a. Since three major mitogen-activated protein kinase (MAPK) pathways have important roles in apoptosis, we examined if Homer 1a is involved in the effects of MAPK pathways on apoptosis. It was shown that inhibition of the ERK1/2 pathway increased the expression and the protective effect of Homer 1a, but inhibition of the p38 pathway produced the opposite effect. Cross-talk among MAPK pathways was also associated with the regulation of Homer 1a during apoptotic cell death. Blocking the p38 pathway increased the activity in the ERK1/2 pathway, while inhibition of ERK1/2 pathway abolished the effect of p38 inhibitor on Homer 1a. Furthermore, Homer 1a reversely affected the activation of MAPK pathways. These findings suggest that Homer 1a plays an important role in the prevention of apoptotic cell death and contributes to distinct regulatory effects of MAPK pathways on apoptotic cell death.     

Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Homer proteins, a new member of the postsynaptic scaffolding proteins, regulate glutamatergic signaling and intracellular calcium mobilization in the central nervous system. Here we investigated the effects of down-regulating Homer1b/c, a constitutively expressed long form of Homer proteins, on glutamate excitotoxicity-induced neuronal injury. In our in vitro excitotoxic models, we demonstrated that glutamate insults led to a dose-dependent neuronal injury, which was mediated by the intracellular calcium-dependent reactive oxygen species (ROS) production. We found that down-regulation of Homer1b/c with specific small interfering RNA (siRNA) improved neuronal survival, inhibited intracellular ROS production, and reduced apoptotic cell death after neurotoxicity. Homer1b/c knockdown decreased the intracellular calcium overload through inhibition of the group I metabotropic glutamate receptor (mGluR)/inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) in injured neurons. In addition, Homer1b/c siRNA transfection attenuated the activation of eukaryotic initiation factor 2α (eIF2α), RNA-dependent protein kinase-like ER kinase (PERK) and caspase-12, and inhibited the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) after glutamate treatment. Homer1b/c knockdown also preserved the mitochondrial membrane potential (MMP), reduced cytochrome c (Cyt. c) release, and partly blocked the increase of capase-9 activity and Bax/Bcl-2 ratio. Taken together, these results suggest that down-regulation of Homer1b/c protects cortical neurons against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the inhibition of calcium-dependent ROS production and the preservation of the ER and mitochondrial function.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.     

Homer proteins accelerate Ca2+ clearance mediated by the plasma membrane Ca2+ pump in hippocampal neurons

The plasma membrane Ca(2+) ATPase (PMCA) is responsible for maintaining basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and returning small increases in [Ca(2+)](i) back to resting levels. The carboxyl terminus of some PMCA splice variants bind Homer proteins; how binding affects PMCA function is unknown. Here, we examined the effects of altered expression of Homer proteins on PMCA-mediated Ca(2+) clearance from rat hippocampal neurons in culture. The kinetics of PMCA-mediated recovery from the [Ca(2+)](i) increase evoked by a brief train of action potentials was determined in the soma of single neurons using indo-1-based photometry. Exogenous expression of Homer 1a, Homer 1c or Homer 2a did not affect PMCA function. However, shRNA mediated knockdown of Homer 1 slowed PMCA mediated Ca(2+) clearance by 28% relative to cells expressing non-silencing shRNA. The slowed recovery rate in cells expressing Homer 1 shRNA was reversed by expression of a short Homer 2 truncation mutant. These results indicate that constitutively expressed Homer proteins tonically stimulate PMCA function in hippocampal neurons. We propose a model in which binding of short or long Homer proteins to the carboxyl terminus of the PMCA stimulates Ca(2+) clearance rate. PMCA-mediated Ca(2+) clearance may be stimulated following incorporation of the pump into Homer organized signaling domains and following induction of the Homer 1a immediate early gene.     

Homer protein increases activation of Ca2+ sparks in permeabilized skeletal muscle

Members of the Homer family of proteins are known to form multimeric complexes capable of cross-linking plasma membrane channels (e.g. metabotropic glutamate receptor) and intracellular Ca2+ release channels (e.g. inositol trisphosphate receptor) in neurons, which potentiates Ca2+ release. Recent work has demonstrated direct interaction of Homer proteins with type 1 and type 2 ryanodine receptor (RyR) isoforms. Moreover, Homer proteins have been shown to modulate RyR-dependent Ca2+ release in isolated channels as well as in whole cell preparations. We now show that long and short forms of Homer H1 (H1c and H1-EVH1) are potent activators of Ca2+ release via RyR in skeletal muscle fibers (e.g. Ca2+ sparks) and potent modulators of ryanodine binding to membranes enriched with RyR, with H1c being significantly more potent than H1-EVH1. Homer did not significantly alter the spatio-temporal properties of the sparks, demonstrating that Homer increases the rate of opening of RyRs, with no change in the overall RyR channel open time and amount of Ca2+ released during a spark. No changes in Ca2+ spark frequency or properties were observed using a full-length H1c with mutation in the EVH1 binding domain (H1c-G89N). One novel finding with each Homer agonist (H1c and H1-EVH1) was that in combination their actions on [3H]ryanodine binding was additive, an effect also observed for these Homer agonists in the Ca2+ spark studies. Finally, in Ca2+ spark studies, excess H1c-G89N prevented the effects of H1c in a dominant negative manner. Taken together our results suggest that the EVH1 domain is critical for the agonist behavior on Ca2+ sparks and ryanodine binding, and that the coiled-coil domain, present in long but not short form Homer, confers an increase in agonist potential apparently through the multimeric association of Homer ligand.     

Glial cell line-derived neurotrophic factor receptor-α1 expressed in striatum in trans regulates development and injury response of dopamine neurons of the substantia nigra

Many of the cellular effects of glial cell line-derived neurotrophic factor are initiated by binding to GNDF family receptor alpha-1 (GFRα1), and mediated by diverse intracellular signaling pathways, most notably through the Ret tyrosine kinase. Ret may be activated by the cell autonomous expression of GFRα1 ('in cis'), or by its non-cell autonomous presence ('in trans'), in either a soluble or immobilized state. GFRα1 is expressed in the striatum, a target of the dopaminergic projection of the substantia nigra. To determine whether post-synaptic expression of GFRα1 in striatum in trans has effects on the development or adult responses to injury of dopamine neurons, we have created transgenic mice in which GFRα1 expression is selectively increased in striatum and other forebrain targets of the dopaminergic projection. Post-synaptic GFRα1 has profound effects on the development of dopamine neurons, resulting in a 40% increase in their adult number. This morphologic effect was associated with an augmented motor response to amphetamine. In adult mice, post-synaptic GFRα1 expression did not affect neuron survival following neurotoxic lesion, but it did increase the preservation of striatal dopaminergic innervation. We conclude that post-synaptic striatal GFRα1 expression has important effects on the biology of dopamine neurons in vivo.     

Human immunodeficiency virus-1 Tat protein and methamphetamine interact synergistically to impair striatal dopaminergic function

The human immunodeficiency virus (HIV)-1 transactivating protein Tat may be pathogenically relevant in HIV-1-induced neuronal injury. The abuse of methamphetamine (MA), which is associated with behaviors that may transmit HIV-1, may damage dopaminergic afferents to the striatum. Since Tat and MA share common mechanisms of injury, we examined whether co-exposure to these toxins would lead to enhanced dopaminergic toxicity. Animals were treated with either saline, a threshold dose of MA, a threshold concentration of Tat injected directly into the striatum, or striatal injections of Tat followed by exposure to MA. Threshold was defined as the highest concentration of toxin that would not result in a significant loss of striatal dopamine levels. One week later, MA-treated animals demonstrated a 7% decline in striatal dopamine levels while Tat-treated animals showed an 8% reduction. Exposure to both MA + Tat caused an almost 65% reduction in striatal dopamine. This same treatment caused a 56% reduction in the binding capacity to the dopamine transporter. Using human fetal neurons, enhanced toxicity was also observed when cells were exposed to both Tat and MA. Mitochondrial membrane potential was disrupted and could be prevented by treatment with antioxidants. This study demonstrates that the HIV-1 'virotoxin' Tat enhances MA-induced striatal damage and suggests that HIV-1-infected individuals who abuse MA may be at increased risk of basal ganglia dysfunction.     

The localization of GABA-Transaminase in the striato-nigral system

The localization of gamma-aminobutyric acid transaminase (GABA-T), the degrading enzyme for γ-aminobutyric acid, was examined in the striatum and substantia nigra using biochemical techniques. Selective destruction of the nigrostriatal dopaminergic system with 6-hydroxydopamine had no effect on the activity of GABA-T in either the striatum or the substantia nigra, although striatal tyrosine hydroxylase activity was reduced by half. Intrastriatal injection of kainic acid in adult rats resulted in a significant dose-dependent decrease in GABA-T activity in both the striatum and the substantia nigra. The decrease in both of these regions was significantly correlated with the decrease in the GABA synthetic enzyme glutamate decarboxylase (GAD). The intrastriatal injection of kainic acid in ten day old rats did not affect striatal GAD or GABA-T activities, although striatal choline acetyl-transferase activity was reduced by half.It is concluded that the GABA-T activity in the striatum is predominantly localized in neuronal elements, although not, apparently, in cholinergic neurons. Some GABA-T activity is also present in the terminals of the striatonigral neurons. However, the dopaminergic nigrostriatal neurons do not appear to contain GABA-T. It is suggested that high GABA-T activity may be characteristic of GABA neurons.     

Wild-type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.     

Analysis of the Proenkephalin Second Messenger-Inducible Enhancer in Rat Striatal Cultures

Abstract: We have previously shown that in cell extracts from rat striatum, cyclic AMP response element (CRE) binding protein (CREB), rather than AP-1 proteins, preferentially interacts with the CRE-2 element of the proenkephalin second messenger-inducible enhancer, even under conditions in which AP-1 proteins are highly induced. Here we use primary striatal cultures to permit a more detailed analysis of CRE-2 function and protein binding in relevant neural cell types. By transfection we find that in primary striatal cultures, as in transformed cell lines, the CRE-1 and CRE-2 elements are required for significant induction by cyclic AMP. We report that cyclic AMP induction of the proenkephalin gene in striatal cultures is protein synthesis independent, excluding a role for newly synthesized proteins like c-Fos. We also show that cyclic AMP induces CREB phosphorylation and that phosphorylated CREB interacts strongly with CRE-2 and weakly with CRE-1. The predominant protein bound to CRE-1 is not CREB, however, and remains to be identified. Despite some prior predictions, we do not find a role for c-Fos in cyclic AMP regulation of proenkephalin gene expression in neurons.     

The viral death protein Apoptin interacts with Hippi,the protein interactor of Huntingtin-interacting protein 1

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells. In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen. We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells. Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization. Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif. Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.     

Dopamine induces apoptosis in young, but not in neonatal, neurons via Ca2+-dependent signal

Dopamine signaling plays a major role in regulation of neuronal apoptosis. During the postnatal period, dopamine signaling is known to be dramatically changed in the striatum. However, because it is difficult to culture neurons after birth, little is known about developmental changes in dopamine-mediated apoptosis. To examine such changes, we established the method of primary culture of striatal neurons from 2- to 3-wk-old (young) mice. Dopamine, via D(1)-like receptors, induced apoptosis in young, but not neonatal, striatal neurons, suggesting that the effect of dopamine on apoptosis changed with development. In contrast, although isoproterenol (Iso), a beta-adrenergic receptor agonist, increased cAMP production to a greater degree than dopamine, Iso did not increase apoptosis in striatal neurons from young and neonatal mice, suggesting a minor role of cAMP in dopamine-mediated apoptosis. Next, we examined the effect of dopamine on Ca(2+) signaling. Dopamine, but not Iso, markedly increased intracellular Ca(2+) in striatal neurons from young mice, and Ca(2+)-chelating agents abolished dopamine-induced apoptosis, suggesting that Ca(2+) played a major role in the dopamine-mediated apoptosis pathway. In contrast, dopamine failed to increase intracellular Ca(2+) in neonatal neurons, and the expression of PLC, which can increase intracellular Ca(2+) via D(1)-like receptor activation, was significantly greater in young than in neonatal striatal neurons. These data suggest that the developmental change in dopamine-mediated Ca(2+) signaling was responsible for differences between young and neonatal striatum in induction of apoptosis. Furthermore, the culture of young striatal neurons is feasible and may provide a new tool for developmental studies.     

Homer proteins shape Xenopus optic tectal cell dendritic arbor development in vivo

Considerable evidence suggests that the Homer family of scaffolding proteins contributes to synaptic organization and function. We investigated the role of both Homer 1b, the constitutively expressed, and developmentally regulated form of Homer, and Homer 1a, the activity-induced immediate early gene, in dendritic arbor elaboration and synaptic function of developing Xenopus optic tectal neurons. We expressed exogenous Homer 1a or Homer 1b in developing Xenopus tectal neurons. By collecting in vivo time lapse images of individual, EGFP-labeled and Homer-expressing neurons over 3 days, we found that Homer 1b leads to a significant decrease in dendritic arbor growth rate and arbor size. Synaptic transmission was also altered in developing neurons transfected with Homer 1b. Cells expressing exogenous Homer 1b over 3 days had a significantly greater AMPA to NMDA ratios, and increased AMPA mEPSC frequency. These data suggest that increasing Homer 1b expression increases excitatory synaptic inputs, increases synaptic maturation, and slows dendritic arbor growth rate. Exogenous Homer 1a expression increases AMPA mEPSC frequency, but did not significantly affect tectal cell dendritic arbor development. Changes in the ratio of Homer 1a to Homer 1b may signal the neuron that overall activity levels in the cell have changed, and this in turn could affect protein interactions at the synapse, synaptic transmission, and structural development of the dendritic arbor.     

Inhibition of metabotropic glutamate receptor signaling by the huntingtin-binding protein optineurin

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.     

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental Hypoxic-Ischemic Injury

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.