Li-Hong Long

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol‐linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cβ. The present work was designed to characterize the Ca2+ signals regulated by this phosphatidylinositol‐linked D1 dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol‐linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long‐lasting increase in basal [Ca2+]i in a time‐ and dose‐dependent manner. Stimulation was observable at 0.1 μm and reached the maximal effect at 30 μm. The [Ca2+]i increase induced by 1 μm SKF83959 reached a plateau in 5 ± 2.13 min, an average 96 ± 5.6% increase over control. The sustained elevation of [Ca2+]i was due to both intracellular calcium release and calcium influx. The initial component of Ca2+ increase through release from intracellular stores was necessary for triggering the late component of Ca2+ rise through influx. We further demonstrated that activation of phospholipase Cβ/inositol triphosphate was responsible for SKF83959‐induced Ca2+ release from intracellular stores. Moreover, inhibition of voltage‐operated calcium channel or NMDA receptor‐gated calcium channel strongly attenuated SKF83959‐induced Ca2+ influx, indicating that both voltage‐operated calcium channel and NMDA receptor contribute to phosphatidylinositol‐linked D1 receptor regulation of [Ca2+]i.

Reversal of aging-associated hippocampal synaptic plasticity deficits by reductants via regulation of thiol redox and NMDA receptor function

Deficits in learning and memory accompanied by age‐related neurodegenerative diseases are closely related to the impairment of synaptic plasticity. In this study, we investigated the role of thiol redox status in the modulation of the N‐methyl‐d‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP) in CA1 areas of hippocampal slices. Our results demonstrated that the impaired LTP induced by aging could be reversed by acute administration of reductants that can regulate thiol redox status directly, such as dithiothreitol or β‐mercaptoethanol, but not by classical anti‐oxidants such as vitamin C or trolox. This repair was mediated by the recruitment of aging‐related deficits in NMDAR function induced by these reductants and was mimicked by glutathione, which can restore the age‐associated alterations in endogenous thiol redox status. Moreover, antioxidant prevented but failed to reverse H2O2‐induced impairment of NMDAR‐mediated synaptic plasticity. These results indicate that the restoring of thiol redox status may be a more effective strategy than the scavenging of oxidants in the treatment of pre‐existing oxidative injury in learning and memory.

The flavonoid baicalein promotes NMDA receptor-dependent long-term potentiation and enhances memory.

BACKGROUND AND PURPOSE There is growing interest in the physiological functions of flavonoids, especially in their effects on cognitive function and on neurodegenerative diseases. The aim of the current investigation was to evaluate the role of the flavonoid baicalein in long‐term potentiation (LTP) in the hippocampal CA1 region and cognitive behavioural performance.

Disruption of PICK1 attenuates the function of ASICs and PKC regulation of ASICs

Acid-sensing ion channels (ASICs) extensively exist in both central and peripheral neuronal systems and contribute to many physiological and pathological processes. The protein that interacts with C kinase 1 (PICK1) was cloned as one of the proteins interacting with protein kinase C (PKC) and colocalized with ASIC1 and ASIC2. Here, we used PICK1 knockout (PICK1-KO) C57/BL6 mice together with the whole cell patch clamp, calcium imaging, RT-PCR, Western blot, and immunocytochemistry techniques to explore the possible change in ASICs and the regulatory effects of PKC on ASICs. The results showed that PICK1 played a key role in regulation of ASIC functions. In PICK1-KO mouse cortical neurons, both the amplitude of ASIC currents and elevation of [Ca(2+)](i) mediated by acid were decreased, which were attributable to the decreased expression of ASIC1a and ASIC2a proteins in the plasma membrane. PKC, a partner protein of PICK1, regulated ASIC functions via PICK1. The agonist and antagonist of PKC only altered ASIC currents and acid-induced increase in [Ca(2+)](i) in wild-type, but not in KO mice. In conclusion, our data provided the direct evidence from PICK1-KO mice that a novel target protein, PICK1, would regulate ASIC function and membrane expression in the brain. In addition, PICK1 played the bridge role between PKC and ASICs.

Activation of Phosphatidylinositol-linked Novel D1 Dopamine Receptor Contributes to the Calcium Mobilization in Cultured Rat Prefrontal Cortical Astrocytes

Recent evidences indicate the existence of an atypical D1 dopamine receptor other than traditional D1 dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase Cβ (PLCβ). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D1 dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca2+]i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca2+]i. Application of D1 receptor, but not D2, α1 adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca2+]i rise, indicating that activation of the D1 dopamine receptor was essential for this response. Increase in [Ca2+]i was a two-step process characterized by an initial increase in [Ca2+]i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca2+ entry. Furthermore, SKF83959-stimulated increase in [Ca2+]i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D1 receptor by SKF83959 mediates a dose-dependent mobilization of [Ca2+]i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.

Orexin-A Activates Hypothalamic AMP-Activated Protein Kinase Signaling through a Ca2+-Dependent Mechanism Involving Voltage-Gated L-Type Calcium Channel

Hypothalamic AMP-activated protein kinase (AMPK) and orexins/hypocretins are both involved in the control of feeding behavior, but little is known about the interaction between these two signaling systems. Here, we demonstrated that orexin-A elicited significant activation of AMPK in the arcuate nucleus (ARC) of the hypothalamus by elevating cytosolic free Ca2+ involving extracellular calcium influx. Electrophysiological results revealed that orexin-A increased the L-type calcium current via the orexin receptor–phospholipase C–protein kinase C signaling pathway in ARC neurons that produce neuropeptide Y, an important downstream effector of orexin-A’s orexigenic effect. Furthermore, the L-type calcium channel inhibitor nifedipine attenuated orexin-A–induced AMPK activation in vitro and in vivo. We found that inhibition of AMPK by either compound C (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine) or the ATP-mimetic 9-β-D-arabinofuranoside prevented the appetite-stimulating effect of orexin-A. This action can be mimicked by nifedipine, the blocker of the L-type calcium channel. Our results indicated that orexin-A activates hypothalamic AMPK signaling through a Ca2+-dependent mechanism involving the voltage-gated L-type calcium channel, which may serve as a potential target for regulating feeding behavior.

HPLC and LC-MS analysis of sinomenine and its application in pharmacokinetic studies in rats

Aim:To improve and validate analytical methods based on HPLC and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the quantitative measurement of sinomenine in rat plasma and brain tissue.Methods:The separation of analytes and the internal standard (IS), chloramphenicol, was performed on an Agilent TC-C18 column (250×4.6 mm, 5 μm). Blood samples were measured with a Surveyor photodiode array (PDA) detector at a wavelength of 263 nm. The LCQ DECA XPPlus mass spectrometer was operated in the multiple reactions monitoring mode using positive electrospray ionization, and the transition from the precursor ion (m/z 279) to the product ion (m/z 224) for sinomenine was measured in brain tissue.Results:Measurements were linear over the concentration range of 0.1–100 μg/mL for sinomenine in plasma and over the range of 0.01–5.00 μg/g for sinomenine in brain tissue. The intra- and inter-day variabilities were less than 10% of the relative standard deviation (RSD), and the extraction and recovery of sinomenine was 72.48%–80.26% from plasma and 73.75%–80.26% from brain tissue. The limit of quantification (LOQ) was 0.1 μg/mL for plasma, and 0.01 μg/g for brain tissue. Identification of sinomenine was reproducible at 0.5, 5, and 50 μg/mL in the plasma and at 0.05, 0.50, and 2.00 μg/g in brain tissue. The concentration of sinomenine measured in brain tissue after a single ip dose had a neuroprotective effect on H2O2-induced injury in PC12 cells in vitro.Conclusion:Our methods offered a sensitivity within a wide linear concentration range for sinomenine. These methods were successfully applied to evaluate sinomenine pharmacokinetics over time in rat brain tissue after a single ip dose of 30 mg/kg.

Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins.

Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinsons disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function.

AGE-RELATED SYNAPTIC CHANGES IN THE CA1 STRATUM RADIATUM AND SPATIAL LEARNING IMPAIRMENT IN RATS

1 Age‐related impairments in hippocampus‐dependent spatial learning and memory are not associated with a loss of neurons, but may be related to synaptic changes. In the present study, we analysed the behavioural performance of adult, middle‐aged and old Wistar rats using the Morris water maze, as well as the structure of synapses and the expression of autophosphorylated Ca2+/calmodulin‐dependent protein kinase II at threonine 286 (pThr286‐αCaMKII), a key post‐synaptic protein in the CA1 stratum radiatum, in the same rats. 2 Old Wistar rats showed significant cognitive deficits. Synaptic density, the area of post‐synaptic densities and the total number of synapses in the CA1 stratum radiatum of old rats were significantly decreased compared with adult rats. The decrease in autophosphorylated pThr286‐αCaMKII was age dependent. 3 These findings reveal that age‐related impairments in learning and memory are associated with synaptic atrophy. The decreased expression of pThr286‐CaMKII may result in reduced synaptic function with ageing.

β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy.

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.