p97 regulates GluA1 homomeric AMPA receptor formation and plasma membrane expression.

The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors (AMPARs) mediate the fast excitatory synaptic transmission in the mammalian brain and are important for synaptic plasticity. In particular, the rapid insertion of the GluA1 homomeric (GluA1-homo) AMPARs into the postsynaptic membrane is considered to be critical in the expression of hippocampal CA1 long-term potentiation (LTP), which is important for certain forms of learning and memory. However, how the formation and trafficking of GluA1-homo AMPARs are regulated remains poorly understood. Here, we report that p97 specifically interacts with and promotes the formation of GluA1-homo AMPARs. The association with p97 retains GluA1-homo AMPARs in the intracellular compartment under basal conditions, and its dissociation allows GluA1-homo AMPARs to be rapidly inserted into the postsynaptic membrane shortly after LTP induction. Thus, our results shed lights into the molecular mechanisms by which p97 regulates GluA1-homo AMPARs formation and trafficking, thereby playing a critical role in mediating synaptic plasticity.

Neural circuits underlying a psychotherapeutic regimen for fear disorders.

A psychotherapeutic regimen that uses alternating bilateral sensory stimulation (ABS) has been used to treat post-traumatic stress disorder. However, the neural basis that underlies the long-lasting effect of this treatment-described as eye movement desensitization and reprocessing-has not been identified. Here we describe a neuronal pathway driven by the superior colliculus (SC) that mediates persistent attenuation of fear. We successfully induced a lasting reduction in fear in mice by pairing visual ABS with conditioned stimuli during fear extinction. Among the types of visual stimulation tested, ABS provided the strongest fear-reducing effect and yielded sustained increases in the activities of the SC and mediodorsal thalamus (MD). Optogenetic manipulation revealed that the SC-MD circuit was necessary and sufficient to prevent the return of fear. ABS suppressed the activity of fear-encoding cells and stabilized inhibitory neurotransmission in the basolateral amygdala through a feedforward inhibitory circuit from the MD. Together, these results reveal the neural circuit that underlies an effective strategy for sustainably attenuating traumatic memories.

The Possible Role of Neurobeachin in Extinction of Contextual Fear Memory.

Established fear memory becomes vulnerable to disruption after memory retrieval and extinction; this labile state is critical for inhibiting the return of fear memory. However, the labile state has a very narrow time window after retrieval, and underlying molecular mechanisms are not well known. To that end, we isolated the hippocampus immediately after fear memory retrieval and performed proteomics. We identified Neurobeachin (NBEA), an autism-related regulator of synaptic protein trafficking, to be upregulated after contextual fear memory retrieval. NBEA protein expression was rapid and transient after fear memory retrieval at the synapse. Nbea mRNA was enriched at the synapses, and the rapid induction of NBEA expression was blocked by inhibition of the mammalian target of rapamycin (mTOR)-dependent signaling pathway. Mice with cornu ammonis 1 (CA1)-specific Nbea shRNA knockdown showed normal fear acquisition and contextual fear memory but impaired extinction, suggesting an important role of Nbea in fear memory extinction processes. Consistently, Nbea heterozygotes showed normal fear acquisition and fear memory recall but showed impairment in extinction. Our data suggest that NBEA is necessary either for induction of memory lability or for the physiological process of memory extinction.

LARGE, an intellectual disability-associated protein, regulates AMPA-type glutamate receptor trafficking and memory.

Mutations in the human LARGE gene result in severe intellectual disability and muscular dystrophy. How LARGE mutation leads to intellectual disability, however, is unclear. In our proteomic study, LARGE was found to be a component of the AMPA-type glutamate receptor (AMPA-R) protein complex, a main player for learning and memory in the brain. Here, our functional study of LARGE showed that LARGE at the Golgi apparatus (Golgi) negatively controlled AMPA-R trafficking from the Golgi to the plasma membrane, leading to down-regulated surface and synaptic AMPA-R targeting. In LARGE knockdown mice, long-term potentiation (LTP) was occluded by synaptic AMPA-R overloading, resulting in impaired contextual fear memory. These findings indicate that the fine-tuning of AMPA-R trafficking by LARGE at the Golgi is critical for hippocampus-dependent memory in the brain. Our study thus provides insights into the pathophysiology underlying cognitive deficits in brain disorders associated with intellectual disability.

Phospholipase C-ß1 Hypofunction in the Pathogenesis of Schizophrenia.

Schizophrenia is a mental disorder that is characterized by various abnormal symptoms. Previous studies indicate decreased expression of phospholipase C-ß1 (PLC-ß1) in the brains of patients with schizophrenia. PLC-ß1-null (PLC-ß1(-/-)) mice exhibit multiple endophenotypes of schizophrenia. Furthermore, a study of PLC-ß1 knockdown in the medial prefrontal cortex of mice has shown a specific behavioral deficit, impaired working memory. These results support the notion that disruption of PLC-ß1-linked signaling in the brain is strongly involved in the pathogenesis of schizophrenia. In this review, we broadly investigate recent studies regarding schizophrenia-related behaviors as well as their various clinical and biological correlates in PLC-ß1(-/-) and knockdown mouse models. This will provide a better understanding of the pathological relevance of the altered expression of PLC-ß1 in the brains of patients with schizophrenia. Evidence accumulated will shed light on future in-depth studies, possibly in human subjects.

Optogenetic control of endogenous Ca(2+) channels in vivo.

Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

Long-term potentiation promotes proliferation/survival and neuronal differentiation of neural stem/progenitor cells.

Neural stem cell (NSC) replacement therapy is considered a promising cell replacement therapy for various neurodegenerative diseases. However, the low rate of NSC survival and neurogenesis currently limits its clinical potential. Here, we examined if hippocampal long-term potentiation (LTP), one of the most well characterized forms of synaptic plasticity, promotes neurogenesis by facilitating proliferation/survival and neuronal differentiation of NSCs. We found that the induction of hippocampal LTP significantly facilitates proliferation/survival and neuronal differentiation of both endogenous neural progenitor cells (NPCs) and exogenously transplanted NSCs in the hippocampus in rats. These effects were eliminated by preventing LTP induction by pharmacological blockade of the N-methyl-D-aspartate glutamate receptor (NMDAR) via systemic application of the receptor antagonist, 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Moreover, using a NPC-neuron co-culture system, we were able to demonstrate that the LTP-promoted NPC neurogenesis is at least in part mediated by a LTP-increased neuronal release of brain-derived neurotrophic factor (BDNF) and its consequent activation of tropomysosin receptor kinase B (TrkB) receptors on NSCs. Our results indicate that LTP promotes the neurogenesis of both endogenous and exogenously transplanted NSCs in the brain. The study suggests that pre-conditioning of the host brain receiving area with a LTP-inducing deep brain stimulation protocol prior to NSC transplantation may increase the likelihood of success of using NSC transplantation as an effective cell therapy for various neurodegenerative diseases.

Depletion of GSH in glial cells induces neurotoxicity: relevance to aging and degenerative neurological diseases.

Oxidative stress induced by inhibition of glutathione (GSH) biosynthesis with D,L-buthionine-S,R-sulfoximine (BSO) causes human microglia, human astrocytes, THP-1 cells, and U373 cells to secrete materials toxic to human neuroblastoma SH-SY5Y cells and stimulates them to release TNF-alpha, IL-6, and nitrite ions. The effect is correlated with activation of the inflammatory pathways P38 MAP- kinase, Jun-N-terminal kinase, and NF-kappaB. The effect is reduced by adding to the medium GSH or clotrimazole (CTM), an inhibitor of Ca(2+)-influx through TRPM2 channels. It is also produced by inhibiting TRPM2 protein expression in microglia and astrocytes through introduction of its small inhibitory RNA (siRNA). TRPM2 mRNA is expressed by glial cells but not by SH-SY5Y cells. BSO in the culture medium causes an almost 3-fold increase in [Ca(2+)](i) in microglia and astrocytes over a 24-h period, which is reduced to half by the addition of CTM. The data strongly suggest that inhibiting intracellular GSH synthesis induces a neuroinflammatory response in human microglia and astrocytes, which is linked to Ca(2+) influx through TRPM2 channels. It represents a new model for inducing neuroinflammation and suggests that increasing GSH levels in glial cells may confer neuroprotection in neurodegenerative diseases, such as Alzheimer disease, which have a prominent neuroinflammatory component.

Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain.

BACKGROUND: Transplantation of neural progenitor cells (NPC) constitutes a putative therapeutic maneuver for use in treatment of neurodegenerative diseases. At present, effects of NPC transplantation in Alzheimer's disease (AD) brain are largely unknown and a primary objective of this work was to demonstrate possible efficacy of NPC administration in an animal model of AD. The benefits of transplantation could involve a spectrum of effects including replacement of endogenous neurons or by conferring neuroprotection with enhancement of neurotrophic factors or diminishing levels of neurotoxic agents. Since chronic inflammation is a characteristic property of AD brain, we considered that transplantation of NPC could have particular utility in inhibiting ongoing inflammatory reactivity. We have tested intrahippocampal transplantation of NPC for efficacy in attenuating inflammatory responses and for neuroprotection in beta-amyloid (Abeta1-42) peptide-injected rat hippocampus. METHODS: Spheres of neural progenitor cells were grown from dissociated telencephalon tissue of rat embryos. NPC were infected with lentiviral vector green fluorescent protein (GFP) with subsequent cell transplantation into rat hippocampus previously injected (3 d prior) with Abeta1-42 peptide or PBS control. Immunohistochemical analysis was carried out (7 d post-NPC transplantation, 10 d post-peptide/PBS injection) for GFP, microgliosis (Iba-1 marker), astrogliosis (GFAP marker), neuron viability (MAP-2 marker) and levels of the proinflammatory cytokine, TNF-alpha. RESULTS: Successful infection of cultured NPC with lentiviral vector green fluorescent protein (GFP) was demonstrated prior to cell transplantation into rat hippocampus. In vivo, immunohistochemical staining showed migration of GFP-positive cells, in a region of dentate gyrus between Abeta1-42/PBS injection site and NPC transplantation site, was increased x2.8-fold with Abeta1-42 compared to PBS injection. Double immunostaining in peptide-injected brain indicated GFP association with nestin and GFAP, but not MAP-2. Cell-specific immunostaining showed marked increases in microgliosis and astrogliosis in Abeta1-42-injected brain (respective increases of x4.3- and x4.6-fold compared with PBS injection). NPC transplantation significantly reduced microgliosis (by 38%) but not astrogliosis in peptide-injected hippocampus. The proinflammatory cytokine TNF-alpha was elevated by 6.7-fold (peptide vs PBS injection) with NPC administration attenuating levels of TNF-alpha (by 40%). Peptide-injected brain demonstrated neuronal loss (MAP-2 staining reduced by 45% vs PBS injection) with NPC transplantation effective in conferring neuroprotection (26% recovery of neurons). CONCLUSIONS: These findings indicate efficacy for NPC transplantation in an animal model of AD with effects consistent with cellular actions to attenuate inflammatory reactivity induced by intrahippocampal peptide injection.

Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries.

Excitotoxic neuronal damage caused by overactivation of N-methyl-D-aspartate glutamate receptors (NMDARs) is thought to be a principal cause of neuronal loss after stroke and brain trauma. Here we report that activation of sterol regulatory element binding protein-1 (SREBP-1) transcription factor in affected neurons is an essential step in NMDAR-mediated excitotoxic neuronal death in both in vitro and in vivo models of stroke. The NMDAR-mediated activation of SREBP-1 is a result of increased insulin-induced gene-1 (Insig-1) degradation, which can be inhibited with an Insig-1-derived interference peptide (Indip) that we have developed. Using a focal ischemia model of stroke, we show that systemic administration of Indip not only prevents SREBP-1 activation but also substantially reduces neuronal damage and improves behavioral outcome. Our study suggests that agents that reduce SREBP-1 activation such as Indip may represent a new class of neuroprotective therapeutics against stroke.

Microglial VEGF receptor response is an integral chemotactic component in Alzheimer's disease pathology.

We hypothesize that microglial chemotactic responses to amyloid-beta peptide (Abeta(1-42)) serve as an early and integral component of inflammatory response in Alzheimer's disease (AD) brain. This study reports a receptor for vascular endothelial growth factor (VEGF), termed VEGF-1 (Flt-1), subserves microglial chemotactic responses induced by Abeta(1-42) stimulation, in vivo and in vitro. Expression of Flt-1 was significantly increased in tissue obtained from AD patients [compared with tissue from nondemented (ND) individuals], in Abeta(1-42)-injected rat hippocampus, and in peptide-stimulated human microglia. Single and double immunohistochemical staining demonstrated marked immunoreactivity, for both Flt-1 and its ligand VEGF, in association with microglia and Abeta deposits in AD, but not ND, brain tissue. Functionally, treatment with anti-Flt-1 antibody was highly effective in inhibiting microglial mobility and chemotactic responses measured in vitro using a transwell migration assay. In vivo, transplanted enhanced green fluorescent protein (EGFP)-labeled microglia exhibited Flt-1-dependent chemotaxis induced by peptide injection with anti-Flt-1 effective in blocking migration of cells. Importantly, anti-Flt-1 reduction of microglial mobility was neuroprotective in peptide-injected hippocampus and associated with a significant increase in numbers of viable hippocampal neurons. The results of this study suggest critical functional roles for Flt-1 in mediating microglial chemotactic inflammatory responses which contribute to pathological conditions in AD brain.

N-methyl amine-substituted fluoxetine derivatives: new dopamine transporter inhibitors.

Transport of dopamine (DA) by the dopamine transporter from the synaptic cleft into the presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. The binding of psychostimulants to their recognition sites on the DA transporter leads to an inhibition of DA transport and a subsequent rising of the dopamine contents in the synaptic cleft is ascribed to a mode of psychostimulation. Discovery of dopamine transporter inhibitors would be useful with regard to substituting for cocaine and minimizing its abuse. Recently, a number of fluoxetine analogues were synthesized, especially focusing on the substitution of N-methyl amine group through modifying the structure of the fluoxetine, N-methyl-3-[p-trifluoromethylphenoxy]-3-phenylpropylamine, widely used as an antidepressant. Among them, the pharmacological properties of FD-2, (R)-N-ethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine and FD-4, N-(R)-3-trifluorophenoxy-3-phenylpropane-imidazole with a higher affinity for the DA transporter were characterized in terms of dopamine transporter inhibition expecting for useful cocaine substitutes. Effects of the compounds on [H(3)]dopamine uptake, [I(125)]RTI-55 binding, and DA transporter-associated currents were examined with the ligand binding assays and voltage clamping technique in human embryonic kidney (HEK)-293 cells where the recombinant human DA transporter (hDAT) was stably expressed. Our results showed that (i) fluoxetine was potent in inhibiting both the uptake of [H(3)]DA (IC(50) = 0.21 +/- 0.032 mM, n = 3) and the [I(125)]RTI-55 binding (IC(50) = 0.23 +/- 0.012 mM, n = 10); (ii) N-methyl amine substituted fluoxetine analogues, FD-2 and FD-4 were equally or more potent than fluoxetine itself in terms of inhibition of [H(3)]DA uptake (IC50 FD-2: 0.077 +/- 0.0032 mM (n = 3); FD-4: 0.26 +/- 0.13 mM (n = 3), inhibition of [I(125)]RTI-55 binding, and reduction in DA transporter-associated currents, suggesting that these analogues could be a new class of dopamine transporter inhibitors.

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro.

OBJECTIVE: To investigate the role of extracellular adenosine triphosphatase (ATP) as an inducer of apoptotic cell death in human granulosa cells and to elucidate its underlying mechanism. DESIGN: Prospective study. SETTING: Gynecologic clinic and human reproduction research laboratory. PATIENT(S): Twenty-five patients undergoing IVF or IVF-ET. INTERVENTION(S): ATP treatment of granulosa luteal cells subjected to primary culture. MAIN OUTCOME MEASURE(S): Apoptosis was assessed by the annexin V binding assay and the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The receptor type that binds ATP, thus mediating ATP-induced apoptosis, was determined by using the calcium imaging and patch-clamp techniques. Mitochondrial depolarization was assessed by staining with the membrane potential-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl carbocyanide iodide (JC-1). RESULT(S): ATP elevated [Ca(2+)](i) by mobilizing intracellularly stored Ca(2+). An ionic membrane current evoked by ATP was mediated by the Ca(2+)-activated K(+) channel. ATP induced a mitochondrial depolarization with a concomitant increase in cellular apoptosis. Treatment with hCG eliminated both ATP-induced mitochondrial depolarization and apoptosis. CONCLUSION(S): Extracellular ATP recognized by P(2Y) type purinoceptor triggers apoptosis in human granulosa luteal cells, and the downstream apoptotic cascade may act at least in part through mitochondria. The antiapoptotic effect of hCG has possible clinical implications in the treatment of disorders involving granulosa cell degeneration (such as follicular atresia).

LB50053: a 5-hydroxytrypamine(1a) agent with a high binding affinity and a potency evoking a K(+) current.

The newly synthesized N-substituted derivative of 3-aryl-pyrrolidine LB50053, 2-[4-[3-(4-fluoro)-phenylpyrrolidine-1-yl] - butyl]-1,2- benzisothiazol -3(2H)-one-1,1-dioxide, was studied in receptor-binding assays and in electrophysiological measurements. Competitive binding experiments with various radioligands to the rat fore-brain revealed that the (S)-enantiomer of LB50053 had a high affinity (Ki 4.2 nmol/l) and a high selectivity for 5-HT(1A) receptors as compared with 5-HT(2A), D(1) dopamine, D(2) dopamine, or (alpha(2)-adrenergic receptor. In Xenopus oocytes, where coupling of the 5-HT(1A) receptor to the G protein activated inwardly rectifying K(+) channel 1(GIRK1) was established, (S)-LB50053 evoked an inward K(+) current through GIRK1 in a manner consistent with a partial agonism. The K(d) value deduced from the dose-response relationships of the 5-HT(1A) receptor full agonist 8-OH-2-(di-n-propylamino)-1,2,3,4-tetrahydronaphthalene and (S)-LB50053 according to Waud analysis was 64.60 nmol/l. These results demonstrate that LB50053 is a 5-HT(1A) receptor partial agonist and thus can be used asa therapeutic or pharmacological research tool for 5-HT(1A) receptor mediated events in the future.