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.

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.

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.

Regulation of basal autophagy by transient receptor potential melastatin 7 (TRPM7) channel.

Macroautophagy (hereafter referred to as autophagy) is a catabolic process for the degradation and recycling of cellular components. Autophagy digests intracellular components, recycling material subsequently used for new protein synthesis. The Ca(2+)- and Mg(2+)-permeable transient receptor potential melastatin 7 (TRPM7) channel underlies the constitutive Ca(2+) influx in some cells. Since autophagy is regulated by cytosolic Ca(2+) level, we set out to determine whether Ca(2+) influx through the TRPM7 channel regulates basal autophagy. When TRPM7 channel expression was induced from HEK293 cells in a nutrient-rich condition, LC3-II level increased indicating the increased level of basal autophagy. The effect of TRPM7 channel on basal autophagy was via Ca(2+)/calmodulin-dependent protein kinase kinase ß, and AMP-activated protein kinase pathway. In contrast, the level of basal autophagy was decreased when the endogenous TRPM7 channel in SH-SY5Y cells was down-regulated using short hairpin RNA. Similarly, an inhibitor for TRPM7 channel decreased the level of basal autophagy. In addition, the inhibitory effect of channel inhibitor on basal autophagy was reversed by increasing extracellular Ca(2+)concentration, suggesting that Ca(2+) influx through TRPM7 channel directly links to basal autophagy. Thus, our studies demonstrate the new role of TRPM7 channel-mediated Ca(2+) entry in the regulation of basal autophagy.

O-GlcNAcylation promotes non-amyloidogenic processing of amyloid-ß protein precursor via inhibition of endocytosis from the plasma membrane.

Amyloid-ß protein precursor (AßPP) is transported to the plasma membrane, where it is sequentially cleaved by α-secretase and γ-secretase. This is called non-amyloidogenic pathway since it precludes the production of amyloid-ß (Aß), the main culprit of Alzheimer's disease (AD). Alternatively, once AßPP undergoes clathrin-dependent endocytosis, it can be sequentially cleaved by ß-secretase and γ-secretase at endosomes, producing Aß (amyloidogenic pathway). ß-N-acetylglucosamine (GlcNAc) can be attached to serine/threonine residues of the target proteins. This novel type of O-linked glycosylation is called O-GlcNAcylation mediated by O-GlcNAc transferase (OGT). The removal of GlcNAc is mediated by O-GlcNAcase (OGN). Recently, it is shown that O-GlcNAcylation of AßPP increases the non-amyloidogenic pathway. To investigate the regulatory role for O-GlcNAcylation in AßPP processing, we first tested the effects of inhibitor for OGN, PUGNAc, on AßPP metabolism in HeLa cells stably transfected with Swedish mutant form of AßPP. Increasing O-GlcNAcylated AßPP level increased α-secretase product while decreased ß-secretase products. We found that PUGNAc increased the trafficking rate of AßPP from the trans-Golgi network to the plasma membrane, and selectively decreased the endocytosis rate of AßPP. These events may contribute to the increased AßPP level in the plasma membrane by PUGNAc. Inhibiting clathrin-dependent endocytosis prevented the effect of PUGNAc on Aß, suggesting that the effect of PUGNAc was mainly mediated by decreasing AßPP endocytosis. These results strongly indicate that O-GlcNAcylation promotes the plasma membrane localization of AßPP, which enhances the non-amyloidogenic processing of AßPP. Thus, O-GlcNAcylation of AßPP can be a potential therapeutic strategy for AD.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C ß1 expression.

Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of ß1 and ß3 isoforms of PLC (PLCß1, PLCß3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCß1 expression, we conclude that increased PLCß1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

Increasing Membrane Cholesterol Level Increases the Amyloidogenic Peptide by Enhancing the Expression of Phospholipase C.

Cerebral elevation of 42-residue amyloid ß-peptide (Aß42) triggers neuronal dysfunction in Alzheimer's disease (AD). Even though a number of cholesterol modulating agents have been shown to affect Aß generation, the role of cholesterol in the pathogenesis of AD is not clear yet. Recently, we have shown that increased membrane cholesterol levels downregulates phosphatidylinositol 4,5-bisphosphate (PIP2) via activation of phospholipase C (PLC). In this study, we tested whether membrane cholesterol levels may affect the Aß42 production via changing PIP2 levels. Increasing membrane cholesterol levels decreased PIP2 and increased secreted Aß42. Supplying PIP2, by using a PIP2-carrier system, blocked the effect of cholesterol on Aß42. We also found that cholesterol increased the expressions of ß1 and ß3 PLC isoforms (PLCß1, PLCß3). Silencing the expression of PLCß1 prevented the effects of cholesterol on PIP2 levels as well as on Aß42 production, suggesting that increased membrane cholesterol levels increased secreted Aß42 by downregulating PIP2 via enhancing the expression of PLCß1. Thus, cholesterol metabolism may be linked to Aß42 levels via PLCß1 expression and subsequent changes in PIP2 metabolism.

Modulation of transient receptor potential melastatin related 7 channel by presenilins.

Presenilins (PS1 and PS2) are multifunctional proteins involved in a diverse array of molecular and cellular functions, including proteolysis, development, neurogenesis, synaptic plasticity, ion channel regulation and phospholipid metabolism. Mutations in presenilin genes are responsible for the majority of Familial Alzheimer disease (FAD). Consequently, FAD-associated mutations in genes encoding PS1 or PS2 lead to several key cellular phenotypes, including alterations in proteolysis of ß-amyloid precursor protein (APP) and Ca(2+) entry. The mechanism underlying presenilin (PS)-mediated modulation of Ca(2+) entry remains to be determined. Our previous studies showed that the PS-dependent down-regulation of phosphatidylinositol-4,5-bisphosphate (PIP2) is attributable to the observed Ca(2+) deficits. In this study, we attempted to identify the ion channel that is subject to the PIP2 and PS-dependent modulation. We found that Ca(2+) or Zn(2+) entry via the transient receptor potential melastatin 7 (TRPM7) channel was attenuated by the presence of FAD-associated PS1 mutants, such as ΔE9 and L286V. TRPM7 has been implicated in Mg(2+) homeostasis and embryonic development. The intracellular delivery of PIP2 restored TRPM7-mediated Ca(2+) influx, indicating that the observed deficits in Ca(2+) entry are due to downregulation of PIP2. Conversely, PS1 and PS2 deficiency, previously shown to upregulate PIP2 levels, potentiated TRPM7-mediated Ca(2+) influx. PS-dependent changes in Ca(2+) influx could be neutralized by a TRPM7 channel blocker. Collectively, these results indicate that TRPM7 may underlie the Ca(2+) entry deficits observed in FAD-associated PS mutants and suggest that the normal function of PS involves regulation of TRPM7 through a PIP2-dependent mechanism.