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.

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.

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.

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.

mGluR long-term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum.

mGluR long-term depression (mGluR-LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR-LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor-controlled Ca2+ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high-conducting Ca2+-permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.

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.

Brain region-specific effects of cGMP-dependent kinase II knockout on AMPA receptor trafficking and animal behavior.

Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO hippocampus is increased as a functional compensation for gene deletion, while such compensation is absent in the prefrontal cortex. Thus, there are brain region-specific effects of cGKII KO on AMPAR trafficking, which could affect animal behavior. Here, we show that GluA1 phosphorylation levels differ in various brain regions, and specific behaviors are altered according to region-specific changes in GluA1 phosphorylation. Moreover, we identified distinct regulations of phosphatases in different brain regions, leading to regional heterogeneity of GluA1 phosphorylation in the KO brain. Our work demonstrates region-specific changes in GluA1 phosphorylation in cGKII KO mice and corresponding effects on cognitive performance. We also reveal distinct regulation of phosphatases in different brain region in which region-specific effects of kinase gene KO arise and can selectively alter animal behavior.

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.

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.

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.

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.

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.