Presynaptic HCN channel activity is required for the expression of long-term potentiation at lateral amygdala to basal amygdala synapses.

Recently, we reported that auditory fear conditioning leads to the presynaptic potentiation at lateral amygdala to basal amygdala (LA-BA) synapses that shares the mechanism with high-frequency stimulation (HFS)-induced long-term potentiation (LTP) ex vivo. In the present study, we further examined the molecular mechanisms underlying the HFS-induced presynaptic LTP. We found that a presynaptic elevation of Ca2+ was required for the LTP induction. Interestingly, the blockade of presynaptic but not postsynaptic HCN channels with ZD7288 completely abolished LTP induction. While ZD7288 did not alter basal synaptic transmission, the blocker fully reversed previously established LTP, indicating that HCN channels are also required for the maintenance of LTP. Indeed, HCN3 and HCN4 channels were preferentially localized in the presynaptic boutons of LA afferents. Furthermore, an inhibition of either GABAB receptors or GIRK channels eliminated the inhibitory effect of HCN blockade on the LTP induction. Collectively, we suggest that activation of presynaptic HCN channels may counteract membrane hyperpolarization during tetanic stimulation, and thereby contributes to the presynaptic LTP at LA-BA synapses.

TRAF2 regulates the protein stability of HIPK2.

A nuclear serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of development and DNA damage response. HIPK2 can induce apoptosis under cellular stress conditions and thus its protein level is maintained low by constant proteasomal degradation. In the present study, we present evidence that TNF receptor-associated factor 2 (TRAF2) regulates the protein stability of HIPK2. Overexpression of TRAF2 decreased while its knockdown increased the HIPK2 protein level. The TRAF2-mediated decrease in HIPK2 protein expression was blocked by proteasomal inhibitor. In addition, TRAF2 decreased the protein half-life of HIPK2. We found that HIPK2 and TRAF2 co-immunoprecipitated. Interestingly, the co-immunoprecipitation was reduced while HIPK2 protein level increased following TNFα treatment, suggesting TNFα induced dissociation of TRAF2 from HIPK2 to accumulate HIPK2. Inhibition of HIPK2 partially suppressed TNFα-induced cell death, indicating that the accumulated HIPK2 may contribute to the TNFα-induced cell death. Our results suggest that TRAF2 can regulate proapoptotic function of HIPK2 by promoting proteasomal degradation.

Regulation of habenular G-protein gamma 8 on learning and memory via modulation of the central acetylcholine system.

Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4ß2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.

Auditory fear conditioning facilitates neurotransmitter release at lateral amygdala to basal amygdala synapses.

The lateral amygdala (LA) is a main sensory input site from the cortical and thalamic regions. In turn, LA glutamatergic pyramidal neurons strongly project to the basal amygdala (BA). Although it is well known that auditory fear conditioning involves synaptic potentiation in the LA, it is not clear whether the LA-BA synaptic transmission is modified upon auditory fear conditioning. Here we found that high-frequency stimulation ex vivo resulted in long-term potentiation (LTP) with a concomitant enhancement of neurotransmitter release at LA-BA synapses. Auditory fear conditioning also led to the presynaptic facilitation at LA-BA synapses. Meanwhile, AMPA/NMDA current ratio was not changed upon fear conditioning, excluding the involvement of postsynaptic mechanism. Notably, fear conditioning occluded electrically induced ex vivo LTP in the LA-BA pathway, indicating that the conditioning and electrically induced LTP share common mechanisms. Our findings suggest that the presynaptic potentiation of LA-BA synapses may be involved in fear conditioning.

PARP-1 regulates mouse embryonic neural stem cell proliferation by regulating PDGFRα expression.

PARP-1 is a multifunctional enzyme that regulates DNA repair, chromatin remodeling, inflammation and cell survival. Our previous study revealed that PARP-1 is required for maintaining normal level of neural stem cell proliferation. In the present study, we present evidence indicating that PARP-1 regulates neural stem cell proliferation by upregulating the expression of platelet-derived growth factor receptor α (PDGFRα). PARP-1 knockout neural stem cells exhibited striking downregulation of PDGFRα expression. We found that PARP-1 promotes the transcription of PDGFRα independently of its enzymatic activity. Overexpression of PDGFRα in the PARP-1 knockout neural stem cells reversed the proliferation defect of the knockout cells. Conversely, knockdown or blocking antibody of PDGFRα suppressed the proliferation of neural stem cells. In addition, blockade of PDGFRα increased cell death rate. Consistent with the downregulation of PDGFRα in the absence of PARP-1, PDGF-AA promoted proliferation of wild-type neural stem cells but not that of PARP-1 knockout cells. These results suggest that PARP-1 can control the neural stem cell proliferation by regulating the expression of PDGFRα.

Chronic social defeat stress-induced enhancement of T-type calcium channels increases burst-firing neurons in the ventral subiculum.

The ventral subiculum (vSub), a representative output structure of the hippocampus, serves as a main limbic region in mediating the brain's response to stress. There are three subtypes of subicular pyramidal neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons, located differently along proximal-distal axis. Here, we found that chronic social defeat stress (CSDS) in mice increased the population of SB neurons but decreased RS neurons in the proximal vSub. Specific blockers of T-type calcium channels inhibited the burst firings with a concomitant reduction of afterdepolarization, suggesting that T-type calcium channels underlie the burst-spiking activity. Consistently, CSDS increased both T-type calcium currents and expression of Cav3.1 proteins, a subtype of T-type calcium channels, in the proximal vSub. Therefore, we conclude that CSDS-induced enhancement of Cav3.1 expression increased bursting neuronal population in the vSub, which may contribute to stress-related behaviors.

Chronic social defeat stress increases burst firing of nucleus accumbens-projecting ventral subicular neurons in stress-susceptible mice.

The ventral subiculum (vSub) is the major output structure of the hippocampus and serves as a main limbic region in mediating the brain's response to stress. Previously, we reported that there are three subtypes of vSub neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons and chronic social defeat stress (CSDS) increased SB neurons especially in the proximal vSub. Here, we found that neurons in the proximal vSub projected to the nucleus accumbens (NAc). CSDS significantly increased SB neurons but decreased RS neurons among the NAc-projecting vSub neuronal population. Interestingly, these changes were only apparent in mice susceptible to CSDS, but not in CSDS-resilient ones. Given that ventral hippocampal inputs to the NAc regulate susceptibility to CSDS, the bursting activity of NAc-projecting vSub neurons might be functionally relevant to behavioral susceptibility to CSDS.

Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.

Neurogenesis occurs spontaneously in the subventricular zone (SVZ) of the lateral ventricle in adult rodent brain, but it has long been debated whether there is sufficient adult neurogenesis in human SVZ. Subcallosal zone (SCZ), a posterior continuum of SVZ closely associated with posterior regions of cortical white matter, has also been reported to contain adult neural stem cells (aNSCs) in both rodents and humans. However, little is known whether SCZ-derived aNSC (SCZ-aNSCs) can produce cortical neurons following brain injury. We found that SCZ-aNSCs exhibited limited neuronal differentiation potential in culture and after transplantation in mice. Neuroblasts derived from SCZ initially migrated toward injured cortex regions following brain injury, but later exhibited apoptosis. Overexpression of anti-apoptotic bcl-xL in the SCZ by retroviral infection rescued neuroblasts from cell death in the injured cortex, but neuronal maturation was still limited, resulting in atrophy. In combination with Bcl-xL, infusion of brain-derived neurotropic factor rescued atrophy, and importantly, a subset of such SCZ-aNSCs differentiated and attained morphological and physiological characteristics of mature, excitatory neurons. These results suggest that the combination of anti-apoptotic and neurotrophic factors might enable the use of aNSCs derived from the SCZ in cortical neurogenesis for neural replacement therapy.

Regional differences in acute corticosterone-induced dendritic remodeling in the rat brain and their behavioral consequences.

BACKGROUND: Glucocorticoid released by stressful stimuli elicits various stress responses. Acute treatment with a single dose of corticosterone (CORT; predominant glucocorticoid of rats) alone has previously been shown to trigger anxiety behavior and robust dendritic hypertrophy of neurons in the basolateral amygdala (BLA). Neurons in the medial prefrontal cortex (mPFC) are also known to be highly sensitive to stress and regulate anxiety-like behaviors. Nevertheless, we know less about acute CORT-induced structural changes of other brain regions and their behavioral outcomes. In addition, the temporal profile of acute CORT effects remains to be examined. The current study investigates time course changes of dendritic architectures in the stress vulnerable brain areas, the BLA and mPFC, and their behavioral consequences after acute treatment with a single dose of CORT. RESULTS: Acute CORT treatment produced delayed onset of dendritic remodeling in the opposite direction in the BLA and mPFC with different time courses. Acute CORT induced dendritic hypertrophy of BLA spiny neurons, which was paralleled by heightened anxiety, both peaked 12 days after the treatment. Meanwhile, CORT-induced dendritic atrophy of mPFC pyramidal neurons peaked on day 6, concomitantly with impaired working memory. Both changed dendritic morphologies and altered behavioral outcomes were fully recovered. CONCLUSION: Our results suggest that stress-induced heightened anxiety appears to be a functional consequence of dendritic remodeling of BLA neurons but not that of mPFC. Instead, stress-induced dendritic atrophy of mPFC neurons is relevant to working memory deficit. Therefore, structural changes in the BLA and the mPFC might be specifically associated with distinct behavioral symptoms observed in stress-related mental disorders. Remarkably, stress-induced dendritic remodeling in the BLA as well as mPFC is readily reversible. The related behavioral outcomes also follow the similar time course in a reversible manner. Therefore, further studies on the cellular mechanism for the plasticity of dendrites architecture might provide new insight into the etiological factors for stress-related mental illness such as posttraumatic stress disorder (PTSD).

Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III.

Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson's disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

SIRT1 negatively regulates the protein stability of HIPK2.

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.

Pathogenic effects of a novel mutation (c.664_681del) in KCNQ4 channels associated with auditory pathology.

Hearing loss is a common communication disorder caused by various environmental and genetic factors. Hereditary hearing loss is very heterogeneous, and most of such cases involve sensorineural defects in the auditory pathway. There are currently 57 known autosomal dominant non-syndromic hearing loss (DFNA) loci, and the causative genes have been identified at 22 of these loci. In the present study, we performed a genome-wide linkage analysis in a Korean family segregating autosomal dominant hearing loss. We observed linkage on chromosome 1p34, and at this locus, we detected a novel mutation consisting of an 18 nucleotide deletion in exon 4 of the KCNQ4 gene, which encodes a voltage-gated potassium channel. We carried out a functional in vitro study to analyze the effects of this mutation (c.664_681del) along with two previously described KCNQ4 mutations, p.W276S and p.G285C. Although the c.664_681del mutation is located in the intercellular loop and the two previously described mutations, p.W276S and p.G285C, are located in the pore region, all mutants inhibit normal channel function by a dominant negative effect. Our analysis indicates that the intercellular loop is as significant as the pore region as a potential site of pathogenic effects on KCNQ4 channel function.

PARP-1 regulates the expression of caspase-11.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multifunctional enzyme that regulates DNA repair, cell death and transcription of inflammatory proteins. In the present study, we present evidence that PARP-1 regulates the expression of caspase-11 following lipopolysaccharide (LPS) stimulation. Knockdown of PARP-1 suppressed the LPS-induced expression of caspase-11 at both mRNA and protein levels as well as caspase-11 promoter activity. Importantly, PARP-1 was recruited to the caspase-11 promoter region containing predicted nuclear factor (NF)-κB-binding sites when examined by chromatin immunoprecipitation assay. However, knockdown of PARP-1 did not suppress the expression of caspase-11 induced by interferon-γ that activates signal transducer and activator of transcription 1 but not NF-κB. PARP-1 enzymatic activity was not required for the caspase-11 upregulation since pharmacological inhibitors of PARP-1 did not suppress the induction of caspase-11. Our results suggest that PARP-1, as a transcriptional cofactor for NF-κB, regulates the induction of caspase-11 at a transcriptional level.

HCN channel activity-dependent modulation of inhibitory synaptic transmission in the rat basolateral amygdala.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the central nervous system and play a regulatory role in neuronal excitability. In the present study, we examined a physiological role of HCN channels in the rat basolateral amygdala (BLA). In vitro electrophysiological studies showed that ZD7288 decreased spontaneous inhibitory postsynaptic current (sIPSC) without changing miniature IPSC (mIPSC). HCN channel blockade also attenuated feedback inhibitions in BLA principal neurons. However, blockade of HCN channel had little effects on spontaneous excitatory postsynaptic current (sEPSC) and mEPSC. Therefore, HCN channel appeared to decrease BLA excitability by increasing the action potential-dependent inhibitory control over the BLA principal neurons. Anxiety is reported to be influenced by neuronal excitability in the BLA and inhibitory synaptic transmission is thought to play a pivotal role in regulating overall excitability of the amygdala. As expected, blockade of HCN channels by targeted injection of ZD7288 to the BLA increased anxiety-like behavior under elevated plus maze test. Our results suggest that HCN channel activity can modulate the GABAergic synaptic transmission in the BLA, which in turn control the amygdala-related emotional behaviors such as anxiety.

Amygdala depotentiation and fear extinction.

Auditory fear memory is thought to be maintained by fear conditioning-induced potentiation of synaptic efficacy, which involves enhanced expression of surface AMPA receptor (AMPAR) at excitatory synapses in the lateral amygdala (LA). Depotentiation, reversal of conditioning-induced potentiation, has been proposed as a cellular mechanism for fear extinction; however, a direct link between depotentiation and extinction has not yet been tested. To address this issue, we applied both ex vivo and in vivo approaches to rats in which fear memory had been consolidated. A unique form of depotentiation reversed conditioning-induced potentiation at thalamic input synapses onto the LA (T-LA synapses) ex vivo. Extinction returned the enhanced T-LA synaptic efficacy observed in conditioned rats to baseline and occluded the depotentiation. Consistently, extinction reversed conditioning-induced enhancement of surface expression of AMPAR subunits in LA synaptosomal preparations. A GluR2-derived peptide that blocks regulated AMPAR endocytosis inhibited depotentiation, and microinjection of a cell-permeable form of the peptide into the LA attenuated extinction. Our results are consistent with the use of depotentiation to weaken potentiated synaptic inputs onto the LA during extinction and provide strong evidence that AMPAR removal at excitatory synapses in the LA underlies extinction.

Suppression of caspase-11 expression by histone deacetylase inhibitors.

It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of kappaB, and activation of nuclear factor-kappaB. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.

Defective neurogenesis and schizophrenia-like behavior in PARP-1-deficient mice.

In the current study we present evidence suggesting that PARP-1 regulates neurogenesis and its deficiency may result in schizophrenia-like behavioral deficits in mice. PARP-1 knockout neural stem cells exhibited a marked upregulation of embryonic stem cell phosphatase that can suppress the proliferative signaling of PI3K-Akt and ERK. The suppressed activity of Akt and ERK in the absence of PARP-1 results in the elevation of FOXO1 activity and its downstream target genes p21 and p27, leading to the inhibition of neural stem cell proliferation. Moreover, expression of neurogenic factors and neuronal differentiation were decreased in the PARP-1 knockout neural stem cells whereas glial differentiation was increased. In accordance with the in vitro data, PARP-1 knockout mice exhibited reduced brain weight with enlarged ventricle as well as decreased adult neurogenesis in the hippocampus. Interestingly, PARP-1 knockout mice exhibited schizophrenia-like symptoms such as anxiety, depression, social interaction deficits, cognitive impairments, and prepulse inhibition deficits. Taken together, our results suggest that PARP-1 regulates neurogenesis during development and in adult and its absence may lead to the schizophrenia-like behavioral abnormality in mice.

Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage.

Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.

Superoxide dismutase 1 mutants related to amyotrophic lateral sclerosis induce endoplasmic stress in neuro2a cells.

One of the common features of damaged neurons in many neurodegenerative diseases is the presence of abnormal aggregates of the disease-related proteins. In amyotrophic lateral sclerosis (ALS) of both sporadic and familial forms, protein aggregates are found in the affected spinal cords. In familial ALS with mutations in copper-zinc superoxide dismutase 1 (SOD1), the propensity of SOD1 for aggregation is known to increase with the mutation. In the present study, we examined whether the aggregate-prone SOD1 mutants induce endoplasmic reticulum (ER) stress and the inhibition of the ER stress protects the cells. The ALS-related mutant G85R SOD1 and G93A SOD1 formed visible aggregates and caused cell death possibly by apoptosis when over-expressed in neuro2a cells. Interestingly, the rate of the mutant SOD1-induced cell death was greater than that of the visible aggregate formation. Expression of the mutant SOD1 caused signs of both early and late ER stress responses, namely, RNA-dependent protein kinase-like ER kinase and eukaryotic initiation factor alpha phosphorylation, Jun amino-terminal kinase activation, activating transcription factor 6-translocation, X-box binding protein 1 mRNA splicing, and caspase 12 activation. The X-box binding protein 1 mRNA splicing activation was also detected in the mutant SOD1-expressing cells even without the visible aggregates. The cell death induced by the mutant SOD1 over-expression looked like apoptosis as evidenced by nuclear morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling. Importantly, an ER stress inhibitor, salubrinal delayed the formation of insoluble aggregates of the mutant SOD1 and suppressed the mutant-induced cell death. In addition, over-expression of the ER-targeted Bcl-xL protected the cells from the mutant SOD1-induced cytotoxicity. These results suggest that the misfolding of ALS-related mutant SOD1 induces ER stress possibly prior to the formation of visible aggregates, which may contribute to the motor neuron degeneration in ALS pathogenesis.

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions.

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.