Ginsenosides Decrease ß-Amyloid Production via Potentiating Capacitative Calcium Entry.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by extracellular amyloid plaques composed of amyloid ß-peptide (Aß). Studies have indicated that Ca2+ dysregulation is involved in AD pathology. It is reported that decreased capacitative Ca2+ entry (CCE), a refilling mechanism of intracellular Ca2+, resulting in increased Aß production. In contrast, constitutive activation of CCE could decrease Aß production. Panax ginseng Meyer is known to enhance memory and cognitive functions in healthy human subjects. We have previously reported that some ginsenosides decrease Aß levels in cultured primary neurons and AD mouse model brains. However, mechanisms involved in the Aß-lowering effect of ginsenosides remain unclear. In this study, we investigated the relationship between CCE and Aß production by examining the effects of various ginsenosides on CCE levels. Aß-lowering ginsenosides such as Rk1, Rg5, and Rg3 potentiated CCE. In contrast, ginsenosides without Aß-lowering effects (Re and Rb2) failed to potentiate CCE. The potentiating effect of ginsenosides on CCE was inhibited by the presence of 2-aminoethoxydipherryl borate (2APB), an inhibitor of CCE. 2APB alone increased Aß42 production. Furthermore, the presence of 2APB prevented the effects of ginsenosides on Aß42 production. Our results indicate that ginsenosides decrease Aß production via potentiating CCE levels, confirming a close relationship between CCE levels and Aß production. Since CCE levels are closely related to Aß production, modulating CCE could be a novel target for AD therapeutics.

O-GlcNAcylation Inhibits Endocytosis of Amyloid Precursor Protein by Decreasing Its Localization in Lipid Raft Microdomains.

Like protein phosphorylation, O-GlcNAcylation is a common post-translational protein modification. We already reported that O-GlcNAcylation of amyloid precursor protein (APP) in response to insulin signaling reduces neurotoxic amyloid-ß (Aß) production via inhibition of APP endocytosis. Internalized APP is delivered to endosomes and lysosomes where Aß is produced. However, the molecular mechanism involved in the effect of APP O-GlcNAcylation on APP trafficking remains unknown. To investigate the relationship between APP O-GlcNAcylation and APP endocytosis, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing APP and BACE1, and cultured rat hippocampal neurons. The present study showed that APP O-GlcNAcylation translocated APP from lipid raft to non-raft microdomains in the plasma membrane by using immunocytochemistry and discontinuous sucrose gradients method. By using the biotinylation method, we also found that APP preferentially underwent endocytosis from lipid rafts and that the amount of internalized APP from lipid rafts was specifically reduced by O-GlcNAcylation. These results indicate that O-GlcNAcylation can regulate lipid raft-dependent APP endocytosis via translocation of APP into non-raft microdomains. Our findings showed a new functional role of O-GlcNAcylation for the regulation of APP trafficking, offering new mechanistic insight for Aß production.

Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains.

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-ß (Aß) is produced via ß-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aß generation. ß-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer's disease.

Substrate-Specific Activation of α-Secretase by 7-Deoxy-Trans-Dihydronarciclasine Increases Non-Amyloidogenic Processing of ß-Amyloid Protein Precursor.

Accumulation of ß-amyloid (Aß) in the brain has been implicated in the pathology of Alzheimer's disease (AD). Aß is produced from the Aß precursor protein (APP) through the amyloidogenic pathway by ß-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aß. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aß production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of Lycoris chejuensis, decreases Aß production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased ß-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver-Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.

High-Cholesterol Diet Decreases the Level of Phosphatidylinositol 4,5-Bisphosphate by Enhancing the Expression of Phospholipase C (PLCß1) in Rat Brain.

Cholesterol is a critical component of eukaryotic membranes, where it contributes to regulating transmembrane signaling, cell-cell interaction, and ion transport. Dysregulation of cholesterol levels in the brain may induce neurodegenerative diseases, such as Alzheimer's disease, Parkinson disease, and Huntington disease. We previously reported that augmenting membrane cholesterol level regulates ion channels by decreasing the level of phosphatidylinositol 4,5-bisphosphate (PIP2), which is closely related to ß-amyloid (Aß) production. In addition, cholesterol enrichment decreased PIP2 levels by increasing the expression of the ß1 isoform of phospholipase C (PLC) in cultured cells. In this study, we examined the effect of a high-cholesterol diet on phospholipase C (PLCß1) expression and PIP2 levels in rat brain. PIP2 levels were decreased in the cerebral cortex in rats on a high-cholesterol diet. Levels of PLCß1 expression correlated with PIP2 levels. However, cholesterol and PIP2 levels were not correlated, suggesting that PIP2 level is regulated by cholesterol via PLCß1 expression in the brain. Thus, there exists cross talk between cholesterol and PIP2 that could contribute to the pathogenesis of neurodegenerative diseases.

O-GlcNAcylation of Amyloid-ß Protein Precursor by Insulin Signaling Reduces Amyloid-ß Production.

Alzheimer's disease (AD) is caused by the accumulation of neurotoxic amyloid-ß (Aß) peptides. Aß is derived from amyloid-ß protein precursor (AßPP). In the non-amyloidogenic pathway, AßPP is cleaved by α-secretase and γ-secretase at the plasma membrane, excluding Aß production. Alternatively, AßPP in the plasma membrane is internalized via endocytosis, and delivered to early endosomes and lysosomes, where it is cleaved by ß-secretase and γ-secretase. Recent studies have shown that insulin in the periphery crosses the blood-brain barrier, and plays important roles in the brain. Furthermore, impaired insulin signaling has been linked to the progression of AD, and intranasal insulin administration improves memory impairments and cognition. However, the underlying molecular mechanisms of insulin treatment remain largely unknown. To investigate the effects of insulin on AßPP processing, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing AßPP, and cultured rat cortical neurons. We found that insulin increased the level of cell surface AßPP, decreasing the endocytosis rate of AßPP. Insulin reduced Aß generation through upregulation of AßPP O-GlcNAcylation via Akt insulin signaling. Our present data suggest that insulin affects Aß production by regulating AßPP processing through AßPP O-GlcNAcylation. These results provide mechanistic insight into the beneficial effects of insulin, and a possible link between insulin deficient diabetes and cerebral amyloidosis in the pathogenesis of AD.

Elevated cellular cholesterol in Familial Alzheimer's presenilin 1 mutation is associated with lipid raft localization of ß-amyloid precursor protein.

Familial Alzheimer's disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of ß-amyloid (Aß) from ß-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aß production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-ß-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts.

Justicidin A Reduces ß-Amyloid via Inhibiting Endocytosis of ß-Amyloid Precursor Protein.

ß-amyloid precursor protein (APP) can be cleaved by α-, and γ-secretase at plasma membrane producing soluble ectodomain fragment (sAPPα). Alternatively, following endocytosis, APP is cleaved by ß-, and γ-secretase at early endosomes generating ß-amyloid (Aß), the main culprit in Alzheimer's disease (AD). Thus, APP endocytosis is critical for Aß production. Recently, we reported that Monsonia angustifolia, the indigenous vegetables consumed in Tanzania, improved cognitive function and decreased Aß production. In this study, we examined the underlying mechanism of justicidin A, the active compound of M. angustifolia, on Aß production. We found that justicidin A reduced endocytosis of APP, increasing sAPPα level, while decreasing Aß level in HeLa cells overexpressing human APP with the Swedish mutation. The effect of justicidin A on Aß production was blocked by endocytosis inhibitors, indicating that the decreased APP endocytosis by justicidin A is the underlying mechanism. Thus, justicidin A, the active compound of M. angustifolia, may be a novel agent for AD treatment.

7-Deoxy-trans-dihydronarciclasine Reduces ß-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer's Disease.

The critical pathological feature of Alzheimer's disease (AD) is the accumulation of ß-amyloid (Aß), the main constituent of amyloid plaques. ß-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by ß- and γ-secretase generating Aß at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aß at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aß accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aß production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aß generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aß, sAPPß, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aß and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.

Polychlorinated biphenyl 19 blocks the most common form of store-operated Ca2+ entry through Orai.

PCB19, a 2,2',6-trichlorinated biphenyl, is one of many non-dioxin-like polychlorinated biphenyls (NDL-PCBs), which are ubiquitous pollutants. NDL-PCBs affect cytosolic Ca2+ signaling by promoting Ca2+ release from ryanodine receptor-sensitive Ca2+ pools and inhibiting store-operated Ca2+ entry (SOCE) from the extracellular space. However, NDL-PCB-mediated SOCE inhibition has only been demonstrated in PC12 cells, in which SOCE is thought to be mainly mediated by TRPC family channels. Here, we investigated the effect of PCB19 on SOCE using human embryonic kidney 293 (HEK293) cells, human leukemia T cell line Jurkat-T cells and human promyelocytoma HL-60 cells which are the cell lines that are previously demonstrated to mediate the most common form of SOCE solely by the intrinsic Orai channels. PCB19 reduced thapsigargin-induced Ca2+ influx after Ca2+ pool depletion in HEK293 cells. SOCEs in HEK293, Jurkat T, HL-60 and PC12 cells showed distinct sensitivities to SOCE inhibitors such as Gd3+ and ML-9; however, PCB19 also showed a common effect of inhibiting SOCEs in all cell lines. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current but not the TRPM7 current. These results imply that PCB19 inhibits not only TRPC-mediated SOCE as in PC12 cells but also Orai-mediated SOCE as in many other cells including HEK293, Jurkat T and HL-60 cells.

Activation of transient receptor potential melastatin 7 (TRPM7) channel increases basal autophagy and reduces amyloid ß-peptide.

Cerebral accumulation of amyloid ß-peptide (Aß), which is produced from amyloid precursor protein (APP), is the primary cause of Alzheimer's disease (AD). Autophagy recycles cellular components and digests intracellular components including Aß. The Ca2+- and Mg2+-permeable transient receptor potential melastatin 7 (TRPM7) channel underlies the constitutive Ca2+ influx in some cells. Since we already reported that TRPM7 channel-mediated Ca2+ influx regulates basal autophagy, we hypothesize that the activation of TRPM7 channel could increase basal autophagy and consequently decrease Aß. In this study, we showed that naltriben (NTB), a specific TRPM7 channel activator, induced Ca2+ influx and activated autophagic signaling in neuroblastoma SH-SY5Y cells. NTB also promoted co-localization of LC3 and APP, and reduced Aß. Furthermore, we found that an early-onset familial AD-associated presenilin1 ΔE9 (PS1 ΔE9) mutant cells had attenuated basal autophagy. NTB was able to recover autophagy and decrease Aß in PS1 ΔE9 cells. Our results show that the activating TRPM7 channel may prevent AD-related Aß neuropathology via modulating Ca2+-regulated basal autophagy.

O-GlcNAcylation of amyloid-ß precursor protein at threonine 576 residue regulates trafficking and processing.

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-ß (Aß) derived from proteolytic processing of amyloid-ß precursor protein (APP). APP undergoes post-translational modification including N- and O-glycosylation. O-GlcNAcylation is a novel type of O-glycosylation, mediated by O-GlcNAc transferase attaching O-ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of the target proteins. O-GlcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GlcNAcylated APP using the O-GlcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aß production. However, O-GlcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GlcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GlcNAcylation levels, indicating that these sites were endogenously O-GlcNAcylated. Thr 576 was the major O-GlcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and Aß production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GlcNAcylation site in APP and indicate that O-GlcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GlcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics.

Protective Roles of Monsonia angustifolia and Its Active Compounds in Experimental Models of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by the accumulation of neurotoxic ß-amyloid (Aß) peptides, which consequently affects cognitive decline and memory impairment. Current research on AD treatment is actively focusing on the prevention of neurotoxic Aß peptide accumulation. Monsonia angustifolia is reported to be consumed as an indigenous vegetable in Tanzania. In this study, we investigated the effect of the ethanol (EtOH) extract of M. angustifolia dried ground material on Aß production and spatial learning ability as protection against AD. The formation of Aß peptides was significantly reduced in HeLa cells stably transfected with the Swedish mutant form of ß-amyloid precursor protein (APPsw) after treatment with a 60% EtOH extract of M. angustifolia. We next examined the cognitive-improving effects of the EtOH extract in vivo. Tg2576 mice were treated with extract for 6 months and subjected to Morris water maze and novel object recognition tests. The results showed that the 60% EtOH extract of M. angustifolia significantly ameliorated behavioral deficits of the AD transgenic mice and reduced the level of insoluble Aß42 in the cerebral cortex and hippocampus. We further found that the 60% EtOH extract was effective for memory function recovery after shorter treatment (4 months). In addition, we isolated and identified several single compounds, justicidin A, 5-methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone, from M. angustifolia and tested these compounds. Among them, justicidin A potently decreased the formation of Aß in APPsw-transfected cells. These data suggest that the 60% EtOH extract of M. angustifolia has the potential to be developed as a treatment of AD. Furthermore, justicidin A may contribute, at least partially, to the Aß alteration observed with the extract treatment.

Bioactive Compound Screen for Pharmacological Enhancers of Apolipoprotein E in Primary Human Astrocytes.

Pharmacological screening in physiologically relevant brain cells is crucial for identifying neuroactive compounds that better translate into in vivo biology and efficacious therapeutics. Pharmacological enhancement of apolipoprotein E (apoE), a cholesterol-transporting apolipoprotein, has been proposed as a promising therapeutic approach for Alzheimer's disease. Several nuclear receptor agonists were initially shown to increase brain apoE levels together with ATP-binding cassette transporter 1 (ABCA1), but their underlying mechanisms remain unclear. To gain an insight on brain apoE regulation, we performed an unbiased high-throughput screening of known drugs and bioactive compounds in cultured human primary astrocytes, the major apoE-producing cell type in the brain. We have identified several small molecules that increase apoE secretion via previously unknown mechanisms, including those not co-inducing ABCA1. These newly identified compounds are active preferentially in human astrocytes but not in an astrocytoma cell line, furnishing new tools for investigating biological pathways underlying brain apoE production.

Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6.

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1ß in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn(-/-) mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cß-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

Stress hormone potentiates Zn(2+)-induced neurotoxicity via TRPM7 channel in dopaminergic neuron.

Zinc toxicity is one of the key factors responsible for the neuronal injuries associated with various neurological conditions. Zinc accumulation in some cells is accompanied by the increase of blood stress hormone levels, which might indicate a functional connection between stress and zinc toxicity. However, the cellular mechanism for the effect of stress on zinc toxicity is not known. Recently, it was reported that the zinc permeable transient receptor potential melastatin 7 (TRPM7) channel may represent a novel target for neurological disorders where zinc toxicity plays an important role. To investigate the effect of stress hormone on zinc-induced cell death, neuroblastoma SH-SY5Y cells were pretreated with urocortin, a corticotropin releasing factor (CRF)-related peptide. Urocortin potentiated zinc-induced cell death at µM range of extracellular zinc concentrations. It significantly increased TRPM7 channel expression, and zinc influx into cytosol. Moreover, application of TRPM7 channel blockers and RNA interference of TRPM7 channel expression attenuated the zinc-induced cell death in urocortin-pretreated cells, indicating that TRPM7 channel may serve as a zinc influx pathway. These results suggest that TRPM7 channel may play a critical role for zinc toxicity associated with stress.

Threonine 576 residue of amyloid-ß precursor protein regulates its trafficking and processing.

Deposition of amyloid-ß (Aß) in the brain is the main culprit of Alzheimer's disease (AD). Aß is derived from sequential proteolytic cleavage of amyloid-ß precursor protein (APP). Newly synthesized APP is transported from endoplasmic reticulum to the plasma membrane via trans-Golgi network (TGN) after post-translational modification including N- and O-glycosylation. APP is internalized through clathrin-dependent endocytosis from the plasma membrane to the early endosomes. In this study, we investigated the regulation of APP trafficking and processing by mutating three threonine residues known as O-glycosylation sites. We separately mutated three threonine residues of APP695 into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. Among these APP mutants, only T576A mutant showed reduced cell surface levels, indicating this residue regulates its trafficking. We also confirmed that trafficking from TGN to the plasma membrane was decreased in T576A mutant. Consistent with these observations, T576A mutant accumulated in the early endosomes, and the secreted Aß level was increased. Thus, these results indicate that threonine 576 residue of APP regulates its trafficking and processing.

Coexistence of glutamatergic spine synapses and shaft synapses in substantia nigra dopamine neurons.

Dopamine neurons of the substantia nigra have long been believed to have multiple aspiny dendrites which receive many glutamatergic synaptic inputs from several regions of the brain. But, here, using high-resolution two-photon confocal microscopy in the mouse brain slices, we found a substantial number of common dendritic spines in the nigral dopamine neurons including thin, mushroom, and stubby types of spines. However, the number of dendritic spines of the dopamine neurons was approximately five times lower than that of CA1 pyramidal neurons. Immunostaining and morphological analysis revealed that glutamatergic shaft synapses were present two times more than spine synapses. Using local two-photon glutamate uncaging techniques, we confirmed that shaft synapses and spine synapses had both AMPA and NMDA receptors, but the AMPA/NMDA current ratios differed. The evoked postsynaptic potentials of spine synapses showed lower amplitudes but longer half-widths than those of shaft synapses. Therefore, we provide the first evidence that the midbrain dopamine neurons have two morphologically and functionally distinct types of glutamatergic synapses, spine synapses and shaft synapses, on the same dendrite. This peculiar organization could be a new basis for unraveling many physiological and pathological functions of the midbrain dopamine neurons.

Effect of Lycoris chejuensis and Its Active Components on Experimental Models of Alzheimer's Disease.

We found that an extract of Lycoris chejuensis and its three isolated active components, narciclasine, 7-deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine, each significantly reduced the formation of amyloid-ß peptides in HeLa cells transfected with an amyloid precursor protein carrying the Swedish mutation up to 45 ± 3.6%. The extract down-regulated amyloid precursor protein, especially the mature form by up to 88%, and reduced the ability of secretases to generate toxic amyloid-ß. Double-transgenic mice treated with the extract for 4 months also showed significantly reduced levels of amyloid-ß and plaques while exhibiting improved memory functions in the Morris water maze and novel object recognition tests. In conclusion, the extract and isolated active components of L. chejuensis decreased the production of amyloid-ß by attenuating amyloid precursor protein levels. Furthermore, the extract improved the disrupted memory functions in animals while inhibiting amyloid plaque formation. Thus, this extract, as well as its active components, could prove beneficial in the treatment of Alzheimer's disease.