Ki Soon Shin

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.

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 α 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.

Resveratrol upregulated heat shock proteins and extended the survival of G93A-SOD1 mice.

In the present study, we investigated whether resveratrol, a SIRT1 activator, can suppress the motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis. Chronic intraperitoneal injection of resveratrol delayed the disease onset and extended survival of the transgenic mice overexpressing G93A-SOD1. The number of surviving motor neurons increased in the resveratrol-injected G93A mice. Importantly, the levels of Hsp25 and Hsp70 were elevated while the level of heat shock factor 1 (HSF1) acetylation decreased in the spinal cords of the resveratrol-injected G93A mice. Our data suggest that resveratrol may protect motor neurons from the mutant SOD1-induced neurotoxicity by promoting SIRT1-mediated deacetylation of HSF1 and subsequent upregulation of Hsps.

The chronic inhibition of nitric oxide synthase enhances cell proliferation in the adult rat hippocampus

We investigated the effect of chronic blocking nitric oxide synthase (NOS), an enzyme producing NO from L-arginine, on granule cell proliferation in the dentate gyrus of adult rats under normal conditions. We treated 7-nitroindazole (7-NI) for 5, 15, and 25 days or N-nitro-L-arginine-methyl ester (L-NAME) for 25 days to block NOS activity and subsequently injected 5-bromo-2-deoxyuridine (BrdU) to detect proliferating cells. The BrdU-immunoreactive (IR) cell number was significantly increased in the 7-NI 15 and 25 day treated group, but not in the control or in the 7-NI 5 day treated group. L-NAME treatment for 25 days significantly increased BrdU-IR cells versus the control and 7-NI 25 day treated group. In addition, nissl staining showed no cell death occurred in the dentate gyrus after 7-NI or L-NAME 25 day treatments. Our results demonstrate that chronic inhibition of NOS increases cell proliferation and has no effect on cell death in the dentate gyrus of the rat hippocampus, which suggests that NO may regulate cell proliferation in the dentate gyrus.

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.

The inhibition of nitric oxide synthase enhances PSA-NCAM expression and CREB phosphorylation in the rat hippocampus.

It is well known that nitric oxide (NO) acts downstream of NMDA receptor activation, which regulates the neural plasticity in the brain. In the present study, the effect of L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor, on neural plasticity in the hippocampus was investigated. L-NAME increased the expression of PSA-NCAM and pCREB in the adult rat hippocampus. The co-localization of PSA-NCAM and pCREB indicates a possible relationship between the two in the granule cell layer in the dentate gyrus. Our results demonstrate that NO, as a subsignal of NMDA receptors, could be involved in the structural plasticity of the granule cell layer in the dentate gyrus by regulating the expression of PSA-NCAM and pCREB in the hippocampus.

GluA1 phosphorylation at serine 831 in the lateral amygdala is required for fear renewal.

Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.

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.

CHIP has a protective role against oxidative stress-induced cell death through specific regulation of endonuclease G.

Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging.

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.