Cheng Che Lee

Insulin promotes dendritic spine and synapse formation by the PI3K/Akt/mTOR and Rac1 signaling pathways.

Insulin and its receptor are broadly expressed throughout the brain and have been postulated to play a crucial role in synaptic plasticity. Although structural remodeling of dendritic spines is associated with stable expression of synaptic plasticity, the role of insulin receptor (IR) signaling in the establishment and dynamic changes of dendritic spines remains unclear. Here we report that insulin promotes dendritic spine formation in primary cultures of rat hippocampal neurons. Conversely, downregulation of IR signaling using a blocking antibody or short hairpin RNAs (shRNAs) resulted in a decrease in number of dendritic spines and caused a significant reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Pharmacological blockade of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway and the small GTPase Rac1 specifically prevented the insulin-induced increase in dendritic spine density. In parallel, genetic ablation of Rac1 expression by lentiviral infection with shRNA abrogated the increase in dendritic spines induced by insulin. More importantly, the increase in dendritic spine density by insulin was accompanied by increasing in presynaptic marker staining density and displayed an increase in mEPSC frequency. Taken together, these results reveal a novel role for IR signaling in the regulation of dendritic spine formation and excitatory synapse development in hippocampal neurons through activation of the PI3K/Akt/mTOR and Rac1 signaling pathways.

Insulin rescues amyloid β-induced impairment of hippocampal long-term potentiation

Cerebral accumulation of amyloid beta-protein (Abeta) is generally believed to play a critical role in the pathogenesis of Alzheimers disease (AD). Recent evidence suggests that Abeta-induced synaptic dysfunction is one of earliest pathogenic events observed in AD. Here we report that synthetic Abeta(1-42) strongly inhibited the induction of long-term potentiation (LTP) in the CA1 region of rat hippocampal slices. To ascertain which Abeta(1-42) sequences contribute to the impairment of LTP, we compared actions of several Abeta fragments and found that the sequence within 25-35 region of Abeta mainly contributes to the expression of LTP impairment. Importantly, we show that insulin and insulin-like growth factor-1 significantly inhibit Abeta oligomer formation, particularly dimers and trimers, and ameliorate the synthetic Abeta-induced suppression of LTP. Furthermore, dithiothreitol was found to be capable of significantly preventing the inhibitory effect of insulin on Abeta oligomer formation. In contrast, hemoglobin promotes Abeta oligomer formation and enhances Abeta-mediated inhibition of LTP induction. These results suggest that insulin may have utility in treating the earliest stages of Abeta-induced synaptic dysfunction in AD patients.

Unconjugated bilirubin exposure impairs hippocampal long-term synaptic plasticity

Background Jaundice is one of the most common problems encountered in newborn infants, due to immaturity of hepatic conjugation and transport processes for bilirubin. Although the majority of neonatal jaundice is benign, some neonates with severe hyperbilirubinemia develop bilirubin encephalopathy or kernicterus. Accumulation of unconjugated bilirubin (UCB) in selected brain regions may result in temporary or permanent impairments of auditory, motor, or cognitive function; however, the molecular mechanisms by which UCB elicits such neurotoxicity are still poorly understood. The present study is undertaken to investigate whether prolonged exposure of rat organotypic hippocampal slice cultures to UCB alters the induction of long-term synaptic plasticity. Methodology/Principal Findings Using electrophysiological recording techniques, we find that exposure of hippocampal slice cultures to clinically relevant concentrations of UCB for 24 or 48 h results in an impairment of CA1 long-term potentiation (LTP) and long-term depression (LTD) induction in a time- and concentration-dependent manner. Hippocampal slice cultures stimulated with UCB show no changes in the secretion profiles of the pro-inflammatory cytokines, interleukin-1β and tumor necrosis factor-α, or the propidium ioide uptake. UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism. Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD. Conclusion/Significance Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus. These observations provide new insights into the molecular mechanisms underlying UCB-induced impairment of hippocampal synaptic plasticity which, in turn, might provide opportunities for the development of novel therapeutic strategies that targets these pathways for treatment.

Repeated Cocaine Administration Decreases 5-HT 2A Receptor-Mediated Serotonergic Enhancement of Synaptic Activity in Rat Medial Prefrontal Cortex

Neural adaptations in the medial prefrontal cortex (mPFC) are thought to be crucial in the development and maintenance of addictive behaviors. The mPFC receives a dense serotonergic (5-hydroxytryptamine, 5-HT) innervation from raphe nuclei and 5-HT exerts complex actions on mPFC pyramidal neurons. The present study, using a rat model of behavioral sensitization to cocaine, was designed to determine whether repeated cocaine exposure in vivo is capable of altering 5-HT-induced regulation of glutamatergic transmission in the mPFC. In layer V pyramidal neurons of the mPFC, application of 5-HT, through activation of 5-HT2A receptors, induced a massive enhancement of spontaneous excitatory postsynaptic currents (sEPSCs). Repeated cocaine administration for 5 days resulted in an attenuation in the ability of 5-HT to enhance sEPSCs. This effect was prevented when cocaine was co-administered with the selective 5-HT2A receptor antagonist ketanserin and was mimicked by repeated 5-HT2A receptor agonist (−)4-iodo-2,5-dimethoxyphenylisopropylamine administration. Repeated cocaine administration is not associated with any changes in the levels of 5-HT2A receptors or regulator of GTP-binding protein signaling 4. These results suggest that cocaine-induced inhibition of 5-HT2A receptor-mediated enhancement of glutamatergic transmission in the mPFC may be caused, at least in part, by the impairment of coupling of 5-HT2A receptors with GTP-binding proteins during cocaine withdrawal. These alterations in 5-HT2A receptor responsiveness in the mPFC may be relevant to the development of behavioral sensitization and withdrawal effects following repeated cocaine administration.

Autophagy-regulated ROS from xanthine oxidase acts as an early effector for triggering late mitochondria-dependent apoptosis in cathepsin S-targeted tumor cells

Cathepsin S (CTSS), which is highly expressed in various malignant tumor cells, has been proposed to promote tumor progression, migration, and invasion. CTSS inhibition not only blocks tumor cell invasion and endothelial tube formation but also induces cellular cytotoxicity. In our previous studies, we have observed that CTSS inhibition induces autophagy, which is responsible for up-regulating xanthine oxidase for early ROS generation and consequent cell death. However, whether the autophagy-regulated early ROS triggers apoptosis remains unclear. We conducted a long-term follow-up study to investigate the relationship between early autophagy and late mitochondria-dependent apoptosis. We demonstrated that early ROS generation is critical for mitochondria damage and the activation of intrinsic apoptotic pathway. Attenuating the early ROS level diminished later mitochondrial damage and downstream apoptotic signaling. Collectively, mitochondria-dependent apoptosis is regulated by autophagy-regulated early ROS, which serves as an early effector that triggers mitochondrial signaling for late apoptosis. The data emphasize the essential role of autophagy-regulated early ROS in triggering late apoptotic signaling.

Cocaine Withdrawal Impairs mGluR5-Dependent Long-Term Depression in Nucleus Accumbens Shell Neurons of Both Direct and Indirect Pathways

We previously reported that animals withdrawn from repeated cocaine exposure exhibited a selective deficit in the ability to elicit metabotropic glutamate receptor 5 (mGluR5)-dependent long-term depression (LTD) in the nucleus accumbens (NAc) shell. To determine whether such impairment occurs in the NAc in a cell-type-specific manner, we used bacterial artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of gene regulatory elements for the dopamine D1 receptor (Drd1) or dopamine D2 receptor (Drd2) to identify distinct subpopulations of medium spiny neurons (MSNs). We found that bath application of group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) reliably induced LTD in both NAc shell and core MSNs of wild-type, hemizygous Drd1-eGFP, and Drd2-eGFP mice. Confirming our previous results, cocaine withdrawal selectively impaired DHPG-LTD in NAc shell Drd1-expressing direct and Drd2-expressing indirect pathway MSNs. We also found that the expression of DHPG-LTD in NAc MSNs was not affected by the Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 1-naphthyl acetyl spermine. Furthermore, systemic administration of mGluR5-negative allosteric modulator fenobam before the daily injection of cocaine preserved mGluR5 function and significantly reduced the expression of cocaine-induced behavioral sensitization. These results reveal that withdrawal from repeated cocaine exposure may result in the impairment of NAc mGluR5-LTD in a subregion- but not cell-type-specific manner and suggests that pharmacological antagonism of mGluR5 may represent a potential strategy for reducing cocaine-induced addictive behaviors.

Conditional deletion of Eps8 reduces hippocampal synaptic plasticity and impairs cognitive function.

ABSTRACT Epidermal growth factor receptor substrate 8 (Eps8) is a multifunctional protein involved in actin cytoskeleton regulation and is abundantly expressed in many brain regions. However, the functional significance of Eps8 in the brain has only just begun to be elucidated. Here, we demonstrate that genetic deletion of Eps8 (Eps8−/−) from excitatory neurons leads to impaired performance in a novel object recognition test. Consistently, Eps8−/− mice displayed a deficit in the maintenance of long‐term potentiation in the CA1 region of hippocampal slices, which was rescued by bath application of N‐methyl‐d‐aspartate receptor (NMDAR) antagonist 2‐amino‐5‐phosphonopentanoate. While Eps8−/− mice showed normal basal synaptic transmission, a significant increase in the amplitude and a significantly slower decay kinetic of NMDAR‐mediated excitatory postsynaptic currents (EPSCs) were observed in hippocampal CA1 neurons. Furthermore, a significant increase in the expression of ifenprodil‐sensitive NMDAR‐mediated EPSCs was observed in neurons from Eps8−/− mice compared with those from wild‐type mice. Eps8 deletion led to decreased mature mushroom‐shaped dendritic spine density but increased complexity of basal dendritic trees of hippocampal CA1 pyramidal neurons. These results implicate NMDAR hyperfunction in the cognitive deficits observed in Eps8−/− mice and demonstrate a novel role for Eps8 in regulating hippocampal long‐term synaptic plasticity and cognitive function. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’. HIGHLIGHTSEps8 deletion results in NMDA receptor hyperfunction.Eps8 deletion promotes decay of long‐term potentiation.Eps8 deletion leads to cognitive deficits.

Cathepsin S attenuates endosomal EGFR signalling: A mechanical rationale for the combination of cathepsin S and EGFR tyrosine kinase inhibitors.

EGF-mediated EGFR endocytosis plays a crucial role in the attenuation of EGFR activation by sorting from early endosomes to late endosomes and transporting them into lysosomes for the final proteolytic degradation. We previously observed that cathepsin S (CTSS) inhibition induces tumour cell autophagy through the EGFR-mediated signalling pathway. In this study, we further clarified the relationship between CTSS activities and EGFR signalling regulation. Our results revealed that CTSS can regulate EGFR signalling by facilitating EGF-mediated EGFR degradation. CTSS inhibition delayed the EGFR degradation process and caused EGFR accumulation in the late endosomes at the perinuclear region, which provides spatial compartments for prolonged EGFR and sustained downstream signal transducer and activator of transcription 3 and AKT signalling. Notably, cellular apoptosis was markedly enhanced by combining treatment with the EGFR inhibitor Iressa and CTSS inhibitor 6r. The data not only reveal a biological role of CTSS in EGFR signalling regulation but also evidence a rationale for its clinical evaluation in the combination of CTSS and EGFR tyrosine kinase inhibitors.

The phospholipid-binding protein SESTD1 negatively regulates dendritic spine density by interfering with Rac1-Trio8 signaling pathway

Dendritic spines are actin-rich protrusions from neuronal dendrites that harbor the majority of excitatory synapses. The balance of spine formation and retraction may influence dendritic integrity. While knowledge of the molecular mechanisms that promote dendritic spine formation has accumulated, little is known about the factors that limit spine formation. Here, we show that SESTD1, a phospholipid-binding protein containing a lipid-binding SEC14-like domain and two spectrin-repeat cytoskeleton interaction domains, negatively regulates dendritic spine density in cultured hippocampal neurons. Overexpression of SESTD1 decreases dendritic spine density in neurons by interfering with the interaction between Rac1 and its guanine nucleotide exchange factor (GEF) Trio8. Conversely, knockdown of SESTD1 increases dendritic spine density. Further analysis reveals that the SPEC1 domain-mediated interaction with Rac1 is required for SESTD1 activity toward a decrease in dendritic spine density. Transfection of GEF domain of Trio8 into neurons rescues SESTD1-mediated decrease in dendritic spine density. More importantly, overexpression of SESTD1 results in a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs), whereas SESTD1 knockdown increases the mEPSC frequency. These results suggest that SESTD1 may act as a negative regulator of the Rac1-Trio8 signaling pathway to reduce dendritic spine density and lower excitatory synaptic transmission in hippocampal neurons.