NP-3 Effects of Osthole Microemulsion by Nasal Administration on the Cholinergic Pathway in AD Mice

Objectives:Fructus Cnidii is the dry ripe fruit of Cnidium monnieri(L.)Cuss.,which belongs to the umbelliferous plant.It has long been used in clinic practice and has been found to have pharmacological activity in the central nervous system.Osthole is the main active component of Fructus Cnidii.However,it shows low bioavailability,fast distribution and elimination,and low concentration in the brain when given orally.In this study,we aimed to develop a new dosage form to increase the osthole concentration in the brain and enhance its pharmacological effects in the central nervous system through reducing the dosage while improving the stability and bioavailability.Thus,microemulsion containing osthole was prepared and the effects of osthole microemulsion were examined in the mouse model of Alzheimer’s disease(AD).Methods:On the basis of pseudo-ternary phase diagram,microemulsion was prepared by using polyoxyethlated Cremophor RH40 as emulsifiers,propylene glycol as assistant emulsifiers and ethyl acetate as the oil phase.The particle size and distribution of osthole microemulsion were detected by laser particle size analyzer and transmission election microscope.The content of osthole was determined by UV spectrophotometry.The effects of osthole microemulsion by nasal administration on the learning and memory abilities in scopolamine-treated mice were assessed by Morris water maze and novel object recognition tests.The superoxide dismutase(SOD)activity,glutathione(GSH)levels and malondialdehyde(MDA)contents in the serum were examined to evaluate the oxidant stress.Choline acetyltransferase(ChAT)and acetylcholinesterase(AChE)expression in the olfactory-basal forebrain pathway were detected by immunohistochemical analysis.We also investigated the acetylcholine(ACh)levels and the histological morphology in the brain.Results:The average particle size of 1μg·μL-1 osthole microemulsion was less than 15 nm.It was characterized as spheres under the transmission electron microscopy,and the osthole was completely encapsulated in the microemulsion core.Morris water maze and novel object recognition tests showed that osthole microemulsion improved spatial and object learning and memory in scopolamine-treated mice.Moreover,osthole microemulsion restored the abnormal activity of SOD and increased the levels of MDA and GSH in the serum.Brain immunohistochemistry staining showed that osthole microemulsion up-regulated ChAT expression,while down-regulated AChE in the olfactory-basal forebrain cholinergic pathway.Additionally,the ACh levels and pathological morphology in the brain were also reversed after nasal administration with osthole microemulsion.Conclusion:The 1μg·μL-1 osthole microemulsion is an ideal dosage form with a small particle size,uniform distribution and high permeation.Osthole microemulsion ameliorated memory impairment in scopolamine-teated mice,likely via the olfactory-basal forebrain cholinergic pathway and by reducing oxidative stress.The results implicate the development of intranasal brain targeting drugs as potential treatment of certain central nervous system diseases,including disorders affecting memory such as Alzheimer’s disease.

NP-12 The Phosphodiesterase-4 Inhibitor Roflumilast Reverses Cognition Deficits and Depression-Like Effects via cAMP Signaling-Mediated Neuroprotection in APP/PS1 Transgenic Mice

Phosphodiesterase-4(PDE4)has been demonstrated to be a promising target for treatment of Alzheimer’s disease(AD).Roflumilast(Rof),a potent PDE4 inhibitor,has been approved for treatment of chronic obstructive pulmonary disease(COPD)in humans.Recent studies have shown that Rof improves cognition at doses that do not cause an emetic response,the major side-effect of PDE4 inhibitors.However,the effect of Rof on cognition associated with AD remains largely unknown.Here we examined the effects of Rof on behavioral dysfunction and the related mechanisms in APP/PS1 double transgenic mice,a widely used model for AD.Mice at 10 months of age were first tested in novel object recognition for memory.The recognition index in APP/PS1 mice was decreased compared to WT mice,which was reversed by Rof at 5 and 10 mg·kg-1.This was then verified in the Morris water-maze test.The escape latency during acquisition training was significantly longer and the entries into the target quadrant during the probe trial were much less compared to WT controls,these were also reversed by Rof.In the tail-suspension and forced-swimming tests,which measure depression-like behavior,APP/PS1 mice showed prolonged immobility time,which was reversed by Rof.In addition,the staining of HE and Nissl showed that Rof reduced the loss of neurons and neurocyte apoptosis in APP/PS1 mice.It also reversed the decreased ratio of Bcl-2/BAX and inhibited the increased expression of PDE4D in the cerebral cortex and hippocampus of APP/PS1 mice.Finally,Rof reversed the decreased levels of cAMP and expression of phosphorylated cAMP response element-binding protein(CREB)and brain derived neurotrophic factor(BDNF)in APP/PS1 mice.Overall,these results suggest that Rof not only improves learning and memory,but attenuates depression-like behavior in AD mice,likely via PDE4D/cAMP/CREB/BDNF signaling-mediated neuroprotection.Therefore,Rof can be a therapeutic agent for AD,in particular the comorbidity of memory deficits and depression.

磷酸二酯酶参与认知与情绪调节的研究进展

磷酸二酯酶(PDE)催化水解cAMP和cGMP,是细胞内降解cAMP和cGMP的唯一途径。PDE是一个多基因大家族酶,包含11型不同家族,它们的结构,分布以及调节方式对抑制剂的敏感性都不同。PDE选择性抑制剂可通过抑制cAMP或cGMP水解来调节学习记忆障碍等中枢神经系统疾病。因此,PDE被认为在中枢神经系统疾病的治疗上具有重要地位。本综述介绍目前PDE参与学习记忆障碍这一中枢神经系统疾病调节的研究进展,而且PDE作为中枢神经系统疾病的治疗靶点,研究其选择性抑制剂具有重要的意义。

NP-17 Icariin Facilitates Amyloid-βRemoval in Astrocytes via increased Autophagy by Up-Regulating Sirt6 Expression

Background:Amyloid-β(Aβ)metabolic imbalance is the pivotal pathogenesis leading to Alzheimer’s disease(AD).In sporadic AD,decreased clearance of Aβimportantly contributes to the onset and progression.Astrocytes,the most abundant cell type in the brain,are mainly responsible for maintaining neuronal homeostasis.Most recently,it has been demonstrated that astrocytes play an important role in regulating Aβmetabolism.Icariin(ICA),a flavonoid glucoside extracted from the traditional Chinese herb Epimedium brevicornu,has been shown to produce protective effects against AD by decreasing Aβproduction.However,it remains unclear whether ICA regulates cellular Aβclearance in the astrocytes.Objective:To examine the regulatory effects of ICA on Aβremoval by astrocytes and explore the mechanisms of its actions.Methods:Uptake and subsequent degradation of Aβby astrocytes were evaluated using a combination of enzyme-linked immunosorbent assay(ELISA)and laser confocal microscopy.The effects of oligomer Aβ(oAβ1-42)and/or ICA on the expressions of sirt6 in the primary astrocytes were examined using western blotting and q-PCR assays.The expression of autophagy markers including P62 and LC3-Ⅱ,and phosphorylated-mTOR were measured by western blotting.In order to determine whether sirt6 is involved in the intracellular Aβmetabolism,sirt6 was knocked down using lentiviral vectors containing sirt6-shRNAs and autophagy levels were assessed by western blotting.Results:①In primary astrocytes,ICA not only significantly increased Aβinternalization but also obviously accelerated its degradation in a concentration-dependent manner.②Treatment of astrocytes with Aβ1-42 at 1μmol·L-1 significantly down-regulated the expression of sirt6,which was rescued by ICA.In addition,ICA at 20μmol·L-1 significantly increased the expression of LC3-Ⅱand markedly decreased the expression of P62 and phosphorylated-mTOR in primary astrocytes.③Sirt6 knockdown in primary astrocytes resulted in decreased cellular Aβuptake and degradation.Simultaneously,silencing of sirt6 in astrocytes increased P62 levels and reduced LC3-Ⅱand phosphorylated-mTOR levels.Conclusion:Taken together,our results demonstrate that sirt6 plays an important role in Aβmetabolism in astrocytes via induction of autophagy.ICA is a potential drug for treatment of AD as it upregulates cellular sirt6.

S2A-5 Targeting PDE4 for Alzheimer’s Disease and Alcoholism:An implication in Alcohol-Related Dementia?

Background:Phosphodiesterase 4(PDE4),one of the 11 PDE enzyme families that hydrolyze cyclic nucleotides,is critical for controlling intracellular cyclic AMP(cAMP)concentrations and plays an important role in regulating alcohol consumption and mediating memory in dementia such as Alzheimer’s disease(AD).Chronic alcohol consumption can cause alcohol-related dementia and 50%~75%of detoxified alcoholics have memory or cognition impairment.However,the role of PDE4 and its mechanism remain to be characterized and elucidated.Methods:Using the water-maze,passive avoidance,or novel object recognition test,we examined the effects of rolipram,a prototypical PDE4 inhibitor,and roflumilast,a potent PDE4 inhibitor which has been approved for treatment of chronic obstructive pulmonary disease(COPD)in humans,on memory loss in APP/PS1 double transgenic mice,a widely used model for AD.In addition,we tested the effects of the PDE4 inhibitors,via ip,intra-gastric,or intrastriatum infusion,on ethanol intake and preference using the mouse two-bottle choice paradigm.Mice deficient in PDE4A,PDE4B,or PDE4D(4AKO,4BKO,and 4DKO,respectively)and their wild type(WT)controls were tested for ethanol consumption and memory;the latter was measured in the absence or presence of beta-amyloid peptide 1-42(Abeta42)infused into the dorsal hippocampus.Results:Similar to rolipram,roflumilast reversed memory deficits in APP/PS1 mice in all the memory tests and reduced ethanol intake and preference in C57BL/6 mice in two-bottle choice.Consistent with the results in the memory tests,roflumilast reduced the loss of neurons and neurocyte apoptosis in AD mice,as shown using HE and Nissl staining.It also reversed the decreased ratio of Bcl-2/BAX in the cerebral cortex and hippocampus of AD mice.In addition,roflumilast reversed the decreased levels of cAMP and expression of phosphorylated cAMP response element-binding protein(CREB)and brain derived neurotrophic factor(BDNF)in AD mice.Compared to the WT controls,4AKO mice displayed significant decreases in ethanol intake and preference and reversal of Abeta42-induced memory deficits.In contrast,4BKO mice only mimicked the ability of 4AKO mice to reduce alcohol consumption while 4DKO mice only to reverse Abeta42-induced memory deficits.In addition,levels of cAMP and phospho-CREB(pCREB)were increased in the hippocampus of 4AKO or 4DKO mice,which also showed reversal of Abeta42-induced decreases in pCREB.Conclusions:These data suggest that PDE4 inhibitors such as roflumilast improve learning and memory in AD and reduce ethanol intake and preference likely via cAMP/CREB/BDNF signaling-mediated neuroprotection.PDE4 isoforms have different roles in mediating ethanol-drinking behavior and memory in AD.The results indicate PDE4A as a potential new target for alcohol-related dementia,although studies with animal models of alcoholrelated dementia are needed to clarify this.

S1-6 PDE4 Inhibitor Ameliorates Neuropathic Pain by Upregulating Spinal Connexin 43 Expression

Peripheral nerve injury downregulates connexin43(Cx43)expression in spinal astrocytes.Therefore,restoration of spinal astrocyte Cx43 expression to normal levels could lead to the reduction of nerve injury-induced pain.It has been shown that inhibitors of phosphodiesterase-4(PDE4),an enzyme catalyzing the hydrolysis of cyclic AMP(cAMP),reverse mechanical pain in mice with neuropathic pain.However,the antinociceptive mechanism remains unclear.In the present study,we evaluated the antinociceptive effect of PDE4 inhibitors and demonstrated a novel mechanism by which PDE4 mediates nociception via Cx43 in spinal astrocytes.Methods:The effects of PDE4 inhibitors on neuropathic pain and Cx43 expression in spinal astrocytes were evaluated in mice with partial sciatic nerve ligation(PSNL).Results:Single or repeated,intraperitoneal treatment with the selective PDE4 inhibitor rolipram or roflumilast of mice with PSNL significantly reduced mechanical hypersensitivity.This was mimicked by intrathecal administration of rolipram or roflumilast.In addition,repeated intrathecal treatment with rolipram or roflumilast of mice with PSNL completely prevented the downregulation of Cx43 expression in the spinal dorsal horn.Conclusion:The results suggest that PDE4 inhibitors ameliorate neuropathic pain,which is mediated by a spinal mechanism through the restoration of spinal Cx43 expression.

S4B-4 A Novel Allosteric Phosphodiesterase 4D Inhibitor BPN14770 Reverses Cognitive Impairment in Humanized PDE4D Mice

Classical inhibitors of PDE4 lack subtype selectivity due to exact amino acid sequence conservation of the catalytic site,and consequently,development of these drugs has stalled due to dose-limiting side effects of nausea and emesis.While use of subtype-selective inhibitors(i.e.,for PDE4A,B,or D)could overcome this issue,conservation of the catalytic region,to which classical inhibitors bind,limits this approach.The present study examined the effects of BPN14770,an allosteric inhibitor of PDE4D,which binds to a primate-specific,N-terminal region,conferring greater than 260-fold selectivity for PDE4D.BPN14770 was 100-fold more potent for improving memory and cognition in humanized PDE4D(hPDE4D)mice,which expressed the primate-specific binding sequence,compared to wild-type mice;meanwhile,it exhibited low potency in a mouse surrogate model for emesis.The behavioral and matching neurochemical data presented established a relationship between PDE4D target engagement and effects on cognition for BPN14770.Furthermore,BPN14770 reversed memory and cognitive deficits induced byβ-amyloid peptide 1-42(Aβ42)in Morris water maze,Y maze and novel object recognition tests in the humanized PDE4D mice.The morphological analyses suggested that the number of dendrites and the dendritic length in the CA1 of hippocampus were significantly increased after the Aβ42-treated hPDE4D mice were administered of BPN14770 for two weeks.The neurochemical and molecular biological assays suggested that neuroplasticity-related proteins and neurotrophic factor BDNF in the hippocampus of hPDE4D mice were significantly increased after the hPDE4D mice were treated with BPN14770.These findings suggest clinical potential for PDE4D selective inhibitors in disorders with cognitive deficits such as Alzheimer’s disease,which affects approximately 20 million people worldwide and nearly 5 million people in the United States.