Co-activation of selective nicotinic acetylcholine receptor subtypes is required to reverse hippocampal network dysfunction and prevent fear memory loss in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia with no known cause and cure. Research suggests that a reduction of GABAergic inhibitory interneurons' activity in the hippocampus by beta-amyloid peptide (Aß) is a crucial trigger for cognitive impairment in AD via hyperexcitability. Therefore, enhancing hippocampal inhibition is thought to be protective against AD. However, hippocampal inhibitory cells are highly diverse, and these distinct interneuron subtypes differentially regulate hippocampal inhibitory circuits and cognitive processes. Moreover, Aß unlikely affects all subtypes of inhibitory interneurons in the hippocampus equally. Hence, identifying the affected interneuron subtypes in AD to enhance hippocampal inhibition optimally is conceptually and practically challenging. We have previously found that Aß selectively binds to two of the three major hippocampal nicotinic acetylcholine receptor (nAChR) subtypes, α7- and α4ß2-nAChRs, but not α3ß4-nAChRs, and inhibits these two receptors in cultured hippocampal inhibitory interneurons to decrease their activity, leading to hyperexcitation and synaptic dysfunction in excitatory neurons. We have also revealed that co-activation of α7- and α4ß2-nAChRs is required to reverse the Aß-induced adverse effects in hippocampal excitatory neurons. Here, we discover that α7- and α4ß2-nAChRs predominantly control the nicotinic cholinergic signaling and neuronal activity in hippocampal parvalbumin-positive (PV+) and somatostatin-positive (SST+) inhibitory interneurons, respectively. Furthermore, we reveal that co-activation of these receptors is necessary to reverse hippocampal network dysfunction and fear memory loss in the amyloid pathology model mice. We thus suggest that co-activation of PV+ and SST+ cells is a novel strategy to reverse hippocampal dysfunction and cognitive decline in AD.

Prenatal exposure to valproic acid reduces synaptic δ-catenin levels and disrupts ultrasonic vocalization in neonates.

Valproic acid (VPA) is an effective and commonly prescribed drug for epilepsy and bipolar disorder. However, children born from mothers treated with VPA during pregnancy exhibit an increased incidence of autism spectrum disorder (ASD). Although VPA may impair brain development at the cellular level, the mechanism of VPA-induced ASD has not been completely addressed. A previous study has found that VPA treatment strongly reduces δ-catenin mRNA levels in cultured human neurons. δ-catenin is important for the control of glutamatergic synapses and is strongly associated with ASD. VPA inhibits dendritic morphogenesis in developing neurons, an effect that is also found in neurons lacking δ-catenin expression. We thus hypothesize that prenatal exposure to VPA significantly reduces δ-catenin levels in the brain, which impairs glutamatergic synapses to cause ASD. Here, we found that prenatal exposure to VPA markedly reduced δ-catenin levels in the brain of mouse pups. VPA treatment also impaired dendritic branching in developing mouse cortical neurons, which was partially reversed by elevating δ-catenin expression. Prenatal VPA exposure significantly reduced synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor levels and postsynaptic density 95 (PSD95) in the brain of mouse pups, indicating dysfunctions in glutamatergic synaptic transmission. VPA exposure also significantly altered ultrasonic vocalization (USV) in newly born pups when they were isolated from their nest. Moreover, VPA-exposed pups show impaired hypothalamic response to isolation, which is required to produce animals' USVs following isolation from the nest. Therefore, these results suggest that VPA-induced ASD pathology can be mediated by the loss of δ-catenin functions.

Ketamine's rapid antidepressant effects are mediated by Ca2+-permeable AMPA receptors.

Ketamine is shown to enhance excitatory synaptic drive in multiple brain areas, which is presumed to underlie its rapid antidepressant effects. Moreover, ketamine's therapeutic actions are likely mediated by enhancing neuronal Ca2+ signaling. However, ketamine is a noncompetitive NMDA receptor (NMDAR) antagonist that reduces excitatory synaptic transmission and postsynaptic Ca2+ signaling. Thus, it is a puzzling question how ketamine enhances glutamatergic and Ca2+ activity in neurons to induce rapid antidepressant effects while blocking NMDARs in the hippocampus. Here, we find that ketamine treatment in cultured mouse hippocampal neurons significantly reduces Ca2+ and calcineurin activity to elevate AMPA receptor (AMPAR) subunit GluA1 phosphorylation. This phosphorylation ultimately leads to the expression of Ca2+-Permeable, GluA2-lacking, and GluA1-containing AMPARs (CP-AMPARs). The ketamine-induced expression of CP-AMPARs enhances glutamatergic activity and glutamate receptor plasticity in cultured hippocampal neurons. Moreover, when a sub-anesthetic dose of ketamine is given to mice, it increases synaptic GluA1 levels, but not GluA2, and GluA1 phosphorylation in the hippocampus within 1 hr after treatment. These changes are likely mediated by ketamine-induced reduction of calcineurin activity in the hippocampus. Using the open field and tail suspension tests, we demonstrate that a low dose of ketamine rapidly reduces anxiety-like and depression-like behaviors in both male and female mice. However, when in vivo treatment of a CP-AMPAR antagonist abolishes the ketamine's effects on animals' behaviors. We thus discover that ketamine at the low dose promotes the expression of CP-AMPARs via reduction of calcineurin activity, which in turn enhances synaptic strength to induce rapid antidepressant actions.

The autism-associated loss of δ-catenin functions disrupts social behavior.

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene has been found in autism spectrum disorder (ASD) patients and results in loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we identify that the G34S mutation increases glycogen synthase kinase 3ß (GSK3ß)-dependent δ-catenin degradation to reduce δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, pharmacological inhibition of GSK3ß activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3ß inhibition-induced restoration of normal social behavior in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3ß inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits.

The autism-associated loss of δ-catenin functions disrupts social behaviors.

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene is found in autism spectrum disorder (ASD) patients and induces loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we discover that the G34S mutation generates an additional phosphorylation site for glycogen synthase kinase 3ß (GSK3ß). This promotes δ-catenin degradation and causes the reduction of δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, inhibition of GSK3ß activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3ß inhibition-induced restoration of normal social behaviors in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3ß inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits. Significance Statement: δ-catenin is important for the localization and function of glutamatergic AMPA receptors at synapses in many brain regions. The glycine 34 to serine (G34S) mutation in the δ-catenin gene is found in autism patients and results in the loss of δ-catenin functions. δ-catenin expression is also closely linked to other autism-risk genes involved in synaptic structure and function, further implying that it is important for the autism pathophysiology. Importantly, social dysfunction is a key characteristic of autism. Nonetheless, the links between δ-catenin functions and social behaviors are largely unknown. The significance of the current research is thus predicated on filling this gap by discovering the molecular, cellular, and synaptic underpinnings of the role of δ-catenin in social behaviors.

Prenatal exposure to valproic acid reduces synaptic δ-catenin levels and disrupts ultrasonic vocalization in neonates.

Valproic acid (VPA) is an effective and commonly prescribed drug for epilepsy and bipolar disorder. However, children born from mothers treated with VPA during pregnancy exhibit an increased incidence of autism spectrum disorder (ASD). Although VPA may impair brain development at the cellular level, the mechanism of VPA-induced ASD has not been completely addressed. A previous study has found that VPA treatment strongly reduces δ-catenin mRNA levels in cultured human neurons. δ-catenin is important for the control of glutamatergic synapses and is strongly associated with ASD. VPA inhibits dendritic morphogenesis in developing neurons, an effect that is also found in neurons lacking δ-catenin expression. We thus hypothesize that prenatal exposure to VPA significantly reduces δ-catenin levels in the brain, which impairs glutamatergic synapses to cause ASD. Here, we found that prenatal exposure to VPA markedly reduced δ-catenin levels in the brain of mouse pups. VPA treatment also impaired dendritic branching in developing mouse cortical neurons, which was reversed by elevating δ-catenin expression. Prenatal VPA exposure significantly reduced synaptic AMPA receptor levels and postsynaptic density 95 (PSD95) in the brain of mouse pups, indicating dysfunctions in glutamatergic synaptic transmission. VPA exposure also significantly altered ultrasonic vocalization (USV) in newly born pups when they were isolated from their nest. Moreover, VPA-exposed pups show impaired hypothalamic response to isolation, which is required to produce animals' USVs following isolation from the nest. Therefore, these results suggest that VPA-induced ASD pathology can be mediated by the loss of δ-catenin functions. Highlights: Prenatal exposure of valproic acid (VPA) in mice significantly reduces synaptic δ-catenin protein and AMPA receptor levels in the pups' brains.VPA treatment significantly impairs dendritic branching in cultured cortical neurons, which is reversed by increased δ-catenin expression.VPA exposed pups exhibit impaired communication such as ultrasonic vocalization.Neuronal activation linked to ultrasonic vocalization is absent in VPA-exposed pups.The loss of δ-catenin functions underlies VPA-induced autism spectrum disorder (ASD) in early childhood.

HIV and FIV glycoproteins increase cellular tau pathology via cGMP-dependent kinase II activation.

As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically improves the lifespan of individuals with HIV, many are now entering the prime age when Alzheimer's disease (AD)-like symptoms begin to manifest. It has been shown that hyperphosphorylated tau, a known AD pathological characteristic, is prematurely increased in the brains of HIV-infected individuals as early as in their 30s and that its levels increase with age. This suggests that HIV infection might lead to accelerated AD phenotypes. However, whether HIV infection causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have previously revealed that the viral glycoproteins HIV gp120 and feline immunodeficiency virus (FIV) gp95 induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII; also known as PRKG2) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons to demonstrate that the presence of HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Taken together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.

Injectable immunogel based on polymerized phenylboronic acid and mannan for cancer immunotherapy.

The recent development and prospects of cancer immunotherapy have led to diversification of the types of therapeutic agents used. By simultaneously administering various agents, a more effective therapeutic effect can be expected due to the synergistic effects of multiple therapeutics. In particular, if a substance with adjuvanticity and tumor antigen is delivered at the same time, enhanced cancer immunotherapy can be achieved through high cross-presentation and antigen-presenting cell (APC) maturation. To this end, we developed a polymerized phenylboronic acid (pPBA)-based immunogel for the simultaneous delivery of mannan, which has adjuvanticity and tumor antigen. The immunogel was formed by simple mixing of the polysaccharide mannan with pPBA through the formation of phenylboronic ester between the diol of mannose monomers and phenylboronic acids of pPBA. The immunogel was slowly degraded by hydrolysis to release the loaded tumor antigen. In addition, the released mannan played a key role in both APC maturation in vitro and the upregulation of cross-presentation. Finally, the pPBA-mannan immunogel exhibited a significant anticancer effect in the 4 T1 cell-inoculated mouse model, implying the potential of a codelivery system of antigens and adjuvants for effective cancer immunotherapy.

Phosphorylation of the AMPA receptor subunit GluA1 regulates clathrin-mediated receptor internalization.

Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic upscaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 (also known as GRIA1) and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 serine 845 (S845) predominantly plays a role in receptor internalization, rather than forward trafficking, during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor AP2, which recruits cargo proteins into endocytic clathrin-coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation upon two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.

Assessing Rab5 Activation in Health and Disease.

The endocytic pathway is a system of dynamically communicating vesicles, known as early endosomes, that internalize, sort, and traffic nutrients, trophic factors, and signaling molecules to sites throughout the cell. In all eukaryotic cells, early endosome functions are regulated by Rab5 activity, dependent upon its binding to GTP, whereas Rab5 bound to GDP represents the biologically inactive form. An increasing number of neurodegenerative diseases are associated with endocytic dysfunction and, in the case of Alzheimer's disease (AD) and Down syndrome (DS), an early appearing highly characteristic reflection of endocytic pathway dysfunction is an abnormal enlargement of Rab5 positive endosomes. In AD and DS, endosome enlargement accompanying accelerated endocytosis and fusion, upregulated transcription of endocytosis-related genes, and aberrant signaling by endosomes are caused by pathological Rab5 overactivation. In this chapter, we describe a battery of methods that have been used to assess Rab5 activation in models of AD/DS and are applicable to other cell and animal disease models. These methods include (1) fluorescence recovery after photobleaching (FRAP) assay; (2) quantitative measurement of endosome size by light, fluorescence and electron microscopy; (3) detection of GTP-Rab5 by in situ immunocytochemistry in vitro and ex vivo; (4) immunoprecipitation and GTP-agarose pull-down assay; (5) biochemical detection of Rab5 in endosome-enriched subcellular fractions obtained by OptiPrep™ density gradient centrifugation of mouse brain.

Loss of cGMP-dependent protein kinase II alters ultrasonic vocalizations in mice, a model for speech impairment in human microdeletion 4q21 syndrome.

Chromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, facial dysmorphisms, mental retardation, and absent or delayed speech. One of the key genes in the affected region of the chromosome is PRKG2, which encodes cGMP-dependent protein kinase II (cGKII). Mice lacking cGKII exhibit restricted growth and deficits in learning and memory, as seen in the human syndrome. However, vocalization impairments in these mice have not been determined. The molecular pathway underlying vocalization impairment in humans is not fully understood. Here, we employed cGKII knockout (KO) mice as a model for the human microdeletion syndrome to test whether vocalizations are affected by loss of the PRKG2 gene. Mice emit ultrasonic vocalizations (USVs) to communicate in social situations, stress, and isolation. We thus recorded ultrasonic vocalizations as a model for human speech. We isolated postnatal day 5-7 pups from the nest to record and analyze USVs and found significant differences in vocalizations of KO mice relative to wild-type and heterozygous mutant mice. KO mice produced fewer calls that were shorter duration and higher frequency. Because neuronal activation in the arcuate nucleus in the hypothalamus is important for the production of animal USVs following isolation from the nest, we assessed neuronal activity in the arcuate nucleus of KO pups following isolation. We found significant reduction of neuronal activation in cGKII KO pups after isolation. Taken together, our studies indicate that cGKII is important for neuronal activation in the arcuate nucleus, which significantly contributes to the production of USVs in neonatal mice. We further suggest cGKII KO mice can be a valuable animal model to investigate pathophysiology of human microdeletion 4q21 syndrome.

Phenylboronic acid conjugated to doxorubicin nanocomplexes as an anti-cancer drug delivery system in hepatocellular carcinoma.

Anti-cancer strategies using nanocarrier systems have been explored in a variety of cancers; these systems can easily be incorporated into tumors via the enhanced permeability and retention (EPR) effect leading to enhanced anti-tumor activity while reducing systemic toxicity by specific tumor-targeting. The prognosis of hepatocellular carcinoma (HCC) is extremely poor when the condition is diagnosed at the unresectable stage as treatment options are limited. In order to improve the treatment of cancer and the overall anti-cancer effect, polymerized phenylboronic acid conjugated doxorubicin (pPBA-Dox) nanocomplexes were generated, and conjugated doxorubicin, which is conventionally used in HCC. The nanocomplexes exhibited enhanced anti-tumor activity via tumor-specific targeting in the subcutaneous and orthotopic HCC syngeneic mice tumor model, implying that the nanocomplexes facilitate the targeted Dox delivery to liver cancer in which the sialic acid is over-expressed. Therefore, this study provides insight into the novel targeted therapy using the nanocomplexes for the treatment of HCC.

Selective coactivation of α7- and α4ß2-nicotinic acetylcholine receptors reverses beta-amyloid-induced synaptic dysfunction.

Beta-amyloid (Aß) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aß can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4ß2-, and α3ß4-nAChRs. Aß selectively affects α7- and α4ß2-nAChRs, but not α3ß4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aß affects synaptic function in AD is not completely understood. Here, we showed that Aß associated with α7- and α4ß2-nAChRs but not α3ß4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aß and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aß. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aß. This provides direct molecular evidence for how Aß selectively interacted with α7- and α4ß2-nAChRs, but not α3ß4-nAChRs. Selective coactivation of α7- and α4ß2-nAChRs also sufficiently reversed Aß-induced AMPA receptor dysfunction, including Aß-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4ß2-nAChRs reversed the Aß-induced disruption of long-term potentiation. These findings support a novel mechanism for Aß's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes.

Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer's disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-ß secretase cleaved C-terminal fragment (APP-ßCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-ßCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3ß. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from ß-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.

Drug-loaded titanium dioxide nanoparticle coated with tumor targeting polymer as a sonodynamic chemotherapeutic agent for anti-cancer therapy.

Sonodynamic therapy utilizes ultrasound (US)-responsive generation of reactive oxygen species (ROS) from sonosensitizer, and it is a powerful strategy for anti-cancer treatment in combination with chemotherapy. Herein, we report a precisely designed sonodynamic chemotherapeutics which exhibits US-responsive drug release via ROS generation from co-loaded sono-sensitizer. Doxorubicin (DOX)-coordinated titanium dioxide nanoparticles (TNPs) were encapsulated with polymeric phenyboronic acid (pPBA) via phenylboronic ester bond between pPBA and DOX. Loaded DOX was readily released under US irradiation due to the ROS-cleavable characteristics of phenylboronic ester bond. The size of nanoparticles was around 200 nm, and DOX was released by ROS generated under US irradiation. Tumor targeting by PBA moiety, intracellular ROS generation, and combined therapeutic effect against tumor cells were confirmed in vitro. Finally, we demonstrated high tumor accumulation and efficient tumor growth inhibition in tumor-bearing mice under US irradiation, which revealed potential as a multi-functional agent for sonodynamic chemotherapy.

Co-activation of selective nicotinic acetylcholine receptors is required to reverse beta amyloid-induced Ca2+ hyperexcitation.

Beta-amyloid (Aß) peptide accumulation has long been implicated in the pathogenesis of Alzheimer's disease (AD). Hippocampal network hyperexcitability in the early stages of the disease leads to increased epileptiform activity and eventually cognitive decline. We found that acute application of 250 nM soluble Aß42 oligomers increased Ca2+ activity in hippocampal neurons in parallel with a significant decrease in activity in Aß42-treated interneurons. A potential target of Aß42 is the nicotinic acetylcholine receptor (nAChR). Three major subtypes of nAChRs (α7, α4ß2, and α3ß4) have been reported in the human hippocampus. Simultaneous inhibition of both α7 and α4ß2 nAChRs mimicked the Aß42 effects on both excitatory and inhibitory neurons. However, inhibition of all 3 subtypes showed the opposite effect. Importantly, simultaneous activation of α7 and α4ß2 nAChRs was required to reverse Aß42-induced neuronal hyperexcitation. We suggest co-activation of α7 and α4ß2 nAChRs is required to reverse Aß42-induced Ca2+ hyperexcitation.

Distinct Roles of GluA2-lacking AMPA Receptor Expression in Dopamine D1 or D2 Receptor Neurons in Animal Behavior.

Dopaminergic signaling in the central nervous system regulates several aspects of animal behavior. In the dopaminergic circuits, there are two classes of neurons that can be differentiated by their expression of dopamine receptors, D1 or D2 receptors (D1Rs or D2Rs). Notably, Ca2+-permeable GluA2-lacking glutamate AMPA receptors (CP-AMPARs) are important for gating synaptic plasticity and gene expression in neurons, and their expression particularly in the striatum affects various forms of animal behavior. However, differential effects of GluA2-lacking AMPARs in D1R or D2R neurons on animal behavior have not been addressed. Here, we employed the Cre-Lox recombination system to remove GluA2 selectively in D1R or D2R neurons to express CP-AMPARs and carried out multiple behavior assays. First, the open-field assay revealed that D2R GluA2 knockout (KO) mice showed hypoactivity, while GluA2 KO in D1R neurons had no effect on locomotor activity. We also revealed that D1R GluA2 KO mice showed delayed learning in the accelerating rotarod test compared with control animals, whereas D2R GluA2 KO animals exhibited complete loss of motor learning. In the sociability test, GluA2-lacking AMPAR expression in D1R neurons induced hypersociability, whereas D2R GluA2 KO mice elicited loss of sociability. Both D1R and D2R GluA2 KO mice consumed less food compared with control animals, while D1R GluA2 KO animals showed significantly more weight gain. Finally, D1R GluA2 KO induced anti-depressant effects, while GluA2-lacking AMPAR expression in D2R neurons promoted depression-like behavior. Taken together, GluA2-lacking CP-AMPAR expression in D1R and D2R neurons differentially affects animal behavior.

HIV induces synaptic hyperexcitation via cGMP-dependent protein kinase II activation in the FIV infection model.

Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.

Sucrose withdrawal induces depression and anxiety-like behavior by Kir2.1 upregulation in the nucleus accumbens.

Dieting induces depression and anxiety among other emotional symptoms. Animal models indicate that repeated access to palatable foods such as sugar induces depression and anxiety-like behavior when the food is no longer available. However, the neurobiological mechanisms of how dietary restriction influences mood have not been fully understood. We used the two-bottle sucrose choice paradigm as an overeating and withdrawal model. Withdrawal after lengthy sucrose overeating elicited depression and anxiety-like behavior, which was reversed by sucrose reinstatement. In the nucleus accumbens (NAc) of sucrose withdrawal animals, dopamine levels and cAMP response element binding protein (CREB) activity were significantly reduced, while the inwardly rectifying K+ channel, Kir2.1, was significantly elevated. In addition, overexpression of Kir2.1 selectively in neurons expressing dopamine D1 receptors was sufficient to induce negative mood-linked behavior in the absence of sucrose overeating experience. As elevated K+ channels reduce neuronal excitability, a sucrose withdrawal-induced increase in Kir2.1 expression is able to decrease NAc activity, which provides a cellular basis for depression and anxiety-like behavior in animals.

Lithium increases synaptic GluA2 in hippocampal neurons by elevating the δ-catenin protein.

Lithium (Li+) is a drug widely employed for treating bipolar disorder, however the mechanism of action is not known. Here we study the effects of Li+ in cultured hippocampal neurons on a synaptic complex consisting of δ-catenin, a protein associated with cadherins whose mutation is linked to autism, and GRIP, an AMPA receptor (AMPAR) scaffolding protein, and the AMPAR subunit, GluA2. We show that Li+ elevates the level of δ-catenin in cultured neurons. δ-catenin binds to the ABP and GRIP proteins, which are synaptic scaffolds for GluA2. We show that Li+ increases the levels of GRIP and GluA2, consistent with Li+-induced elevation of δ-catenin. Using GluA2 mutants, we show that the increase in surface level of GluA2 requires GluA2 interaction with GRIP. The amplitude but not the frequency of mEPSCs was also increased by Li+ in cultured hippocampal neurons, confirming a functional effect and consistent with AMPAR stabilization at synapses. Furthermore, animals fed with Li+ show elevated synaptic levels of δ-catenin, GRIP, and GluA2 in the hippocampus, also consistent with the findings in cultured neurons. This work supports a model in which Li+ stabilizes δ-catenin, thus elevating a complex consisting of δ-catenin, GRIP and AMPARs in synapses of hippocampal neurons. Thus, the work suggests a mechanism by which Li+ can alter brain synaptic function that may be relevant to its pharmacologic action in treatment of neurological disease.