Resveratrol Attenuates Aβ<Subscript>25–35</Subscript> Caused Neurotoxicity by Inducing Autophagy Through the TyrRS-PARP1-SIRT1 Signaling Pathway

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Resveratrol (RSV) is a natural polyphenol that has been found to be beneficial for AD through attenuation of Aβ-induced toxicity in neurons both in vivo and in vitro. However, the specific underlying mechanisms remain unknown. Recently, autophagy was found to protect neurons from toxicity injuries via degradation of impaired proteins and organelles. Therefore, the aim of this study was to determine the role of autophagy in the anti-neurotoxicity effect of RSV in PC12 cells. We found that RSV pretreatment suppressed β-amyloid protein fragment 25–35 (Aβ_25–35)-induced decrease in cell viability. Expression of light chain 3-II, degradation of sequestosome 1, and formation of autophagosomes were also upregulated by RSV. Suppression of autophagy by 3-methyladenine abolished the favorable effects of RSV on Aβ_25–35-induced neurotoxicity. Furthermore, RSV promoted the expression of sirtuin 1 (SIRT1), auto-poly-ADP-ribosylation of poly (ADP-ribose) polymerase 1 (PARP1), as well as tyrosyl transfer-RNA (tRNA) synthetase (TyrRS). Nevertheless, RSV-mediated autophagy was markedly abolished with the addition of inhibitors of SIRT1 (EX527), nicotinamide phosphoribosyltransferase (STF-118804), PARP1 (AG-14361), as well as SIRT1 and TyrRS small interfering RNA transfection, indicating that the action of RSV on autophagy induction was dependent on TyrRS, PARP1 and SIRT1. In conclusion, RSV attenuated neurotoxicity caused by Aβ_25–35 through inducing autophagy in PC12 cells, and the autophagy was partially mediated via activation of the TyrRS-PARP1-SIRT1 signaling pathway.     

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.     

Protective Effect of Bajijiasu Against β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-amyloid peptide (Aβ), a major protein component of senile plaques associated with Alzheimer’s disease (AD), is also directly neurotoxic. Mitigation of Aβ-induced neurotoxicity is thus a possible therapeutic approach to delay or prevent onset and progression of AD. This study evaluated the protective effect of Bajijiasu (β- d-fructofuranosyl (2–2) β- d-fructofuranosyl), a dimeric fructose isolated from the Chinese herb Radix Morinda officinalis, on Aβ-induced neurotoxicity in pheochromocytoma (PC12) cells. Bajijiasu alone had no endogenous neurotoxicity up to 200 μM. Brief pretreatment with 10–40 μM Bajijiasu (2 h) significantly reversed the reduction in cell viability induced by subsequent 24 h exposure to Aβ_25–35 (21 μM) as measured by MTT and LDH assays, and reduced Aβ_25–35-induced apoptosis as indicated by reduced annexin V-EGFP staining. Bajijiasu also decreased the accumulation of intracellular reactive oxygen species and the lipid peroxidation product malondialdehyde in PC12 cells, upregulated expression of glutathione reductase and superoxide dismutase, prevented depolarization of the mitochondrial membrane potential (Ψm), and blocked Aβ_25–35-induced increases in [Ca²⁺] _i . Furthermore, Bajijiasu reversed Aβ_25–35-induced changes in the expression levels of p21, CDK4, E2F1, Bax, NF-κB p65, and caspase-3. Bajijiasu is neuroprotective against Aβ_25–35-induced neurotoxicity in PC12 cells, likely by protecting against oxidative stress and ensuing apoptosis.     

Involvement of α7 nAChR Signaling Cascade in Epigallocatechin Gallate Suppression of β-Amyloid-Induced Apoptotic Cortical Neuronal Insults

Excessive generation and accumulation of the β-amyloid (Aβ) peptide in selectively vulnerable brain regions is a key pathogenic event in the Alzheimer's disease (AD), while epigallocatechin gallate (EGCG) is a very promising chemical to suppress a variety of Aβ-induced neurodegenerative disorders. However, the precise molecular mechanism of EGCG responsible for protection against neurotoxicity still remains elusive. To validate and further investigate the possible mechanism involved, we explored whether EGCG neuroprotection against neurotoxicity of Aβ is mediated through the α7 nicotinic acetylcholine receptor (α7 nAChR) signaling cascade. It was shown in rat primary cortical neurons that short-term treatment with EGCG significantly attenuated the neurotoxicity of Aβ_1–42, as demonstrated by increased cell viability, reduced number of apoptotic cells, decreased reactive oxygen species (ROS) generation, and downregulated caspase-3 levels after treatment with 25-μM Aβ_1–42. In addition, EGCG markedly strengthened activation of α7nAChR as well as its downstream pathway signaling molecules phosphatidylinositol 3-kinase (PI3K) and Akt, subsequently leading to suppression of Bcl-2 downregulation in Aβ-treated neurons. Conversely, administration of α7nAChR antagonist methyllycaconitine (MLA; 20 μM) to neuronal cultures significantly attenuated the neuroprotection of EGCG against Aβ-induced neurototoxicity, thus presenting new evidence that the α7nAChR activity together with PI3K/Akt transduction signaling may contribute to the molecular mechanism underlying the neuroprotective effects of EGCG against Aβ-induced cell death.     

Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Abstract

Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ_25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ_25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ_25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.     

Docosahexaenoic acid withstands the Aβ25-35-induced neurotoxicity in SH-SY5Y cells

Background

Docosahexaenoic acid (DHA, C22:6, n-3) ameliorates the memory-related learning deficits of Alzheimer's disease (AD), which is characterized by fibrillar amyloid deposits in the affected brains. Here, we have investigated whether DHA-induced inhibition of Amyloid β-peptide_25-35 (Aβ_25-35) fibrillation limits or deteriorates the toxicity of the human neuroblastoma cells (SH-SY5Y).

Experimental methods

In vitro fibrillation of Aβ_25-35 was performed in the absence or presence of DHA. Afterwards, SH-SY5Y cells were incubated with Aβ_25-35 in absence or presence 20 μM DHA to evaluate its effect on the Aβ_25-35-induced neurotoxicity by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]-redox and TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay and immunohistochemistry. The level of Aβ_25-35-induced lipid peroxide (LPO) was determined in the absence or presence of oligomer-specific antibody. Fatty acid profile was estimated by gas chromatography.

Results

DHA significantly reduced the Aβ_25-35 in vitro fibrillation, as indicated by fluorospectroscopy and transmission electron microscopy. Aβ_25-35 decreased the MTT-redox activity and increased the apoptotic damage and levels of LPO when compared with those of the controls. However, when the SH-SY5Y cells were treated with Aβ_25-35 in the presence of DHA, MTT redox potential significantly increased and the levels LPO decreased, suggesting an inhibition of the Aβ_25-35-induced neurotoxity. DHA improved the Aβ_25-35 induced DNA damage and axodendritic loss, with a concomitant increase in the cellular level of DHA, suggesting DHA protects the cell from neurotoxic degeneration.

Conclusion

DHA not only inhibits the in vitro fibrillation but also resists the Aβ_25-35-induced toxicity in the neuronal cells. This might be the basis of the DHA-induced amelioration of Aβ-induced neurodegeneration and related cognitive deficits.     

Protective Effects of Hesperidin Against Amyloid-β (Aβ) Induced Neurotoxicity Through the Voltage Dependent Anion Channel 1 (VDAC1)-Mediated Mitochondrial Apoptotic Pathway in PC12 Cells

Amyloid-β (Aβ) is known to exert cytotoxic effects by inducing mitochondrial dysfunction. Additionally, the mitochondrial voltage-dependent anion channel 1 (VDAC1), which is involved in the release of apoptotic proteins with possible relevance in Alzheimer’s disease (AD) neuropathology, plays an important role in maintaining mitochondrial function and integrity. However, the application of therapeutic drugs, especially natural products in (AD) therapy via VDAC1-regulated mitochondrial apoptotic pathway has not aroused extensive attention. In the present study, we investigated neuroprotective effects of hesperidin, a bioactive flavonoid compound, on Aβ_25–35-induced neurotoxicity in PC12 cells and also examined the potential cellular signalling mechanism. Our results showed that treatment with hesperidin significantly inhibited Aβ_25–35-induced apoptosis by reversing Aβ-induced mitochondrial dysfunction, including the mitochondrial permeability transition pore opening, intracellular free calcium increase and reactive oxygen species production. Further study indicated that hesperidin can increase the level of VDAC1 phosphorylation through enhancing the activity of the glycogen synthasekinase-3β and decrease the level of hexokinaseI in mitochondrial, resulting in mitochondrial release of cytochrome c. Furthermore, hesperidin inhibited mitochondria-dependent downstream caspase-mediated apoptotic pathway, such as that involving caspase-9 and caspase-3. These results demonstrate that hesperidin can protect Aβ-induced neurotoxicity via VDAC1-regulated mitochondrial apoptotic pathway, and they raise the possibility that hesperidin could be developed into a clinically valuable treatment for AD and other neuronal degenerative diseases associated with mitochondrial dysfunction.     

Neohesperidin Prevents Aβ<Subscript>25–35</Subscript>-Induced Apoptosis in Primary Cultured Hippocampal Neurons by Blocking the S-Nitrosylation of Protein-Disulphide Isomerase

A growing body of literature has established a link between the cerebral ischaemic injury and pathological state of Alzheimer’s disease (AD), and this correlation indicated that the preventive agent for ischaemia might improve the pathology of AD. Our previous studies have demonstrated that Neohesperidin (NH) exhibited neuroprotective effects against cerebral ischemia via the down-regulation of Bcl-2, Akt/PI3K and Nrf2 pathways. In the present study, we first confirmed the protective effects of NH on Aβ_25–35-induced neurotoxicity on primary cultured hippocampal neurons. We further demonstrated NH attenuated Aβ_25–35-induced apoptosis by preventing neurotoxicity associated with lethal UPR and ER stress via blocking S-nitrosylation of protein-disulphide isomerase (PDI). These results suggested that S-nitrosylation of PDI and ER dysfunction might be the synergistic and synchronous pathological process between cerebral ischaemia and AD.     

Deletion of microsomal prostaglandin E synthase-1 protects neuronal cells from cytotoxic effects of β-amyloid peptide fragment 31-35

Epidemiological studies have suggested that the long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity moderates the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E(2) (PGE(2)), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. To examine the involvement in AD pathology of microsomal PGES-1 (mPGES-1), a PGES enzyme, we here prepared primary cerebral neuronal cells from the cerebri of wild-type and mPGES-1-deficient mice and then treated them with β-amyloid (Aβ) fragment 31-35 (Aβ(31-35)), which represents the shortest sequence of native Aβ peptide required for neurotoxicity. Treatment of wild-type neuronal cells with Aβ(31-35) induced mPGES-1 gene expression and PGE(2) production, followed by significant apoptotic cell death, but apoptosis was not induced in mPGES-1-deficient cells. Furthermore, the combined treatment of Aβ(31-35) and PGE(2) induced apoptosis in mPGES-1-deficient neuronal cells. These results indicated that mPGES-1 is induced during Aβ-mediated neuronal cell death and is involved in Aβ-induced neurotoxicity associated with AD pathology.     

5-LOX in Alzheimer’s Disease: Potential Serum Marker and In Vitro Evidences for Rescue of Neurotoxicity by Its Inhibitor YWCS

The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of Alzheimer’s disease (AD) which includes Aβ amyloid deposition and tau hyperphosphorylation. This study determined the level of 5-LOX in serum of AD patients, mild cognitive impairment (MCI) patients, and the normal elderly, and the rescue effect by YWCS, a peptide inhibitor of 5-LOX on neurotoxicity by Aβ amyloid_25–35 (Aβ_25–35) in neuroblastoma cells. The concentration of serum 5-LOX was estimated by surface plasmon resonance and western blot. The neuroprotective effect of 5-LOX peptide inhibitor YWCS in Aβ_25–35-induced neurotoxicity was analyzed by MTT assay and western blotting. We found significant upregulated serum 5-LOX in AD patients and also in MCI patients compared to the normal control group. The peptide inhibitor of 5-LOX, YWCS, prevented the neurotoxic effect of Aβ_25–35 by reducing the expression of γ-secretase as well as p-Tau₁₈₁ in SH-SY5Y cells. However, YWCS was nontoxic towards normal HEK cells. The differential expression of serum 5-LOX among the study groups suggests it can be one of potential serum protein marker and a therapeutic regimen for AD and MCI. The negative correlation with neuropsychological parameters, i.e., MoCA and HMSE, increases its importance and makes it useful during the clinical setup which is very needful in developing countries. Peptide YWCS can serve as a new platform as a 5-LOX inhibitor which can prevent neurotoxicity developed in AD.     

Effect of conjugated linoleic acid, μ-calpain inhibitor,on pathogenesis of Alzheimer's disease

μ-Calpain is a calcium-dependent cysteine protease, which is activated by μM concentration of calcium in vitro. Disrupted intracellular calcium homeostasis leads to hyper-activation of μ-calpain. Hyper-activated μ-calpain enhances the accumulation of β-amyloid peptide by increasing the expression level of β-secretase (BACE1) and induces hyper-phosphorylation of tau along with the formation of neurofibrillary tangle by mediating p35 cleavage into p25, both of which are the major mechanisms of neurodegeneration in Alzheimer's disease (AD). Hence, inhibition of μ-calpain activity is very important in the treatment and prevention of AD. In this study, conjugated linoleic acid (CLA), an eighteen-carbon unsaturated fatty acid, was discovered as a μ-calpain-specific inhibitor. CLA showed neuroprotective effects against neurotoxins such as H₂O₂ and Aβ_1–42 in SH-SY5Y cells, and inhibited Aβ oligomerization/fibrillation and Aβ-induced Zona Occludens-1 degradation. In addition, CLA decreased the levels of proapoptotic proteins, p35 conversion to p25 and tau phosphorylation. These findings implicate CLA as a new core structure for selective μ-calpain inhibitors with neuroprotective effects. CLA should be further evaluated for its potential use as an AD therapeutic agent.     

The emerging role of microRNA-4487/6845-3p in Alzheimer’s disease pathologies is induced by Aβ25–35 triggered in SH-SY5Y cell

Background

Accumulation of amyloid β-peptide (Aβ) is implicated in the pathogenesis and development of Alzheimer’s disease (AD). Neuron-enriched miRNA was aberrantly regulated and may be associated with the pathogenesis of AD. However, regarding whether miRNA is involved in the accumulation of Aβ in AD, the underlying molecule mechanism remains unclear. Therefore, we conduct a systematic identification of the promising role of miRNAs in Aβ deposition, and shed light on the molecular mechanism of target miRNAs underlying SH-SY5Y cells treated with Aβ-induced cytotoxicity.

Results

Statistical analyses of microarray data revealed that 155 significantly upregulated and 50 significantly downregulated miRNAs were found on the basis of log2 | Fold Change |?≥?0.585 and P?< 0.05 filter condition through 2588 kinds of mature miRNA probe examined. PCR results show that the expression change trend of the selected six miRNAs (miR-6845-3p, miR-4487, miR-4534, miR-3622-3p, miR-1233-3p, miR-6760-5p) was consistent with the results of the gene chip. Notably, Aβ_25–35 downregulated hsa-miR-4487 and upregulated hsa-miR-6845-3p in SH-SY5Y cell lines associated with Aβ-mediated pathophysiology. Increase of hsa-miR-4487 could inhibit cells apoptosis, and diminution of hsa-miR-6845-3p could attenuate axon damage mediated by Aβ_25–35 in SH-SY5Y.

Conclusions

Together, these findings suggest that dysregulation of hsa-miR-4487 and hsa-miR-6845-3p contributed to the pathogenesis of AD associated with Aβ25–35 mediated by triggering cell apoptosis and synaptic dysfunction. It might be beneficial to understand the pathogenesis and development of clinical diagnosis and treatment of AD. Further, our well-designed validation studies will test the miRNAs signature as a prognostication tool associated with clinical outcomes in AD.

     

Activation of SIRT1 by curcumin blocks the neurotoxicity of amyloid-β25–35 in rat cortical neurons

As one of the most important hallmarks of Alzheimer’s disease (AD), β-amyloid (Aβ) plays important roles in inducing reactive oxygen species (ROS) generation, mitochondrial dysfunction and apoptotic cell death in neurons. Curcumin extracted from the yellow pigments spice plant turmeric shows multiplied bioactivities such as antioxidant and anti-apoptosis properties in vitro and in vivo. In the present study, the neuroprotective effect of curcumin against Aβ_25–35-induced cell death in cultured cortical neurons was investigated. We found that pretreatment of curcumin prevented the cultured cortical neurons from Aβ_25–35-induced cell toxicity. In addition, curcumin improved mitochondrial membrane potential (ΔΨm), decreased ROS generation and inhibited apoptotic cell death in Aβ_25–35 treated neurons. Furthermore, we found that application of curcumin activated the expression of SIRT1 and subsequently decreased the expression of Bax in the presence of Aβ_25–35. The protective effect of curcumin was blocked by SIRT1 siRNA. Taken together, our results suggest that activation of SIRT1 is involved in the neuroprotective action of curcumin.     

The Neuroprotective Effects of Justicidin A on Amyloid Beta25–35-Induced Neuronal Cell Death Through Inhibition of Tau Hyperphosphorylation and Induction of Autophagy in SH-SY5Y Cells

Justicidin A is a structurally defined arylnaphthalide lignan, which has been shown anti-cancer activity; however, the neuroprotective effect of justicidin A is still untested. In this study, we investigated the action of justicidin A on amyloid beta (Aβ)_25–35-induced neuronal cell death via inhibition of the hyperphosphorylation of tau and induction of autophagy in SH-SY5Y cells. Pretreatment with justicidin A significantly elevated cell viability in cells treated with Aβ_25–35. Western blot data demonstrated that justicidin A inhibited the Aβ_25–35-induced up-regulation the levels of hyperphosphorylation of tau in SH-SY5Y cells. In addition, treatment with justicidin A significantly induced autophagy as measured by the increasing LC3 II/I ratio, an important autophagy marker. These studies showed that justicidin A inhibited activity of glycogen synthase kinase-3beta (GSK-3β), which is an important kinase in up-stream signaling pathways; inhibited hyperphosphorylation of tau in AD; and enhanced activity of AMP-activated protein kinase (AMPK), which is the key molecule for both hyperphosphorylation of tau and induction of autophagy. These data provide the first evidence that justicidin A protects SH-SY5Y cells from Aβ_25–35-induced neuronal cell death through inhibition of hyperphosphorylation of tau and induction of autophagy via regulation the activity of GSK-3β and AMPK, and they also provide some insights into the relationship between tau protein hyperphosphorylation and autophagy. Thus, we conclude that justicidin A may have a potential role for neuroprotection and, therefore, may be used as a therapeutic agent for AD.

     

Protective Effect of Paeoniflorin on Aβ25–35-Induced SH-SY5Y Cell Injury by Preventing Mitochondrial Dysfunction

Alzheimer’s disease (AD) is a major neurodegenerative brain disorder affecting about 14 million people worldwide. Aβ-induced cell injury is a crucial cause of neuronal loss in AD, thus the suppression of which might be useful for the treatment of this disease. In this study, we aimed to evaluate the effect of paeoniflorin (PF), a monoterpene glycoside isolated from aqueous extract of Radix Paeoniae Alba, on Aβ_25–35-induced cytotoxicity in SH-SY5Y cells. The results showed PF could attenuate or restore the viability loss, apoptotic increase, and ROS production induced by Aβ_25–35 in SH-SY5Y cells. In addition, PF strikingly inhibited Aβ_25–35-induced mitochondrial dysfunction, which includes decreased mitochondrial membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and activity of caspase-3 and caspase-9. Therefore, our study provided the first experimental evidence that PF could modulate ROS production and apoptotic mitochondrial pathway in model of neuron injury in vitro and which might provide new insights into its application toward Alzheimer’s disease therapy.     

Thymoquinone Prevents β-Amyloid Neurotoxicity in Primary Cultured Cerebellar Granule Neurons

Thymoquinone (TQ), a bioactive constituent of Nigella sativa Linn (N. sativa) has demonstrated several neuropharmacological attributes. In the present study, the neuroprotective properties of TQ were investigated by studying its anti-apoptotic potential to diminish β-amyloid peptide 1–40 sequence (Aβ_1–40)-induced neuronal cell death in primary cultured cerebellar granule neurons (CGNs). The effects of TQ against Aβ_1–40-induced neurotoxicity, morphological damages, DNA condensation, the generation of reactive oxygen species, and caspase-3, -8, and -9 activation were investigated. Pretreatment of CGNs with TQ (0.1 and 1 μM) and subsequent exposure to 10 μM Aβ_1–40 protected the CGNs against the neurotoxic effects of the latter. In addition, the CGNs were better preserved with intact cell bodies, extensive neurite networks, a loss of condensed chromatin and less free radical generation than those exposed to Aβ_1–40 alone. TQ pretreatment inhibited Aβ_1–40-induced apoptosis of CGNs via both extrinsic and intrinsic caspase pathways. Thus, the findings of this study suggest that TQ may prevent neurotoxicity and Aβ_1–40-induced apoptosis. TQ is, therefore, worth studying further for its potential to reduce the risks of developing Alzheimer’s disease.     

miR-200a-3p promotes β-Amyloid-induced neuronal apoptosis through down-regulation of SIRT1 in Alzheimer’s disease

The aberrantly expressed microRNAs (miRNAs) including miR-200a-3p have been reported in the brains of Alzheimer’s disease (AD) patients in recent researches. Nevertheless, the role of miR-200a-3p in AD has not been characterized. The purpose of this study was to examine whether miR-200a-3p regulated β-Ameyloid (Aβ)-induced neuronal apoptosis by targeting SIRT1, a known anti-apoptotic protein. An increased level of miR-200a-3p and a decreased level of SIRT1 in the hippocampus of APPswe/PSΔE9 mice (a model for AD) were observed. To construct an in vitro cell model of AD, PC12 cells were cultured in presence of Aβ_25-35. The results of flow cytometry analysis showed that the apoptosis rate and cleaved-caspase-3 expression in PC12 cells exposed to Aβ_25-35 were remarkably increased, but the apoptosis rate and cleaved-caspase-3 activity were decreased when cells were transfected with anti-miR-200a-3p. On the other hand, MTT assay showed that the cell survival rate was increased in the Aβ_25-35 + anti-miR-200a-3p group compared with the Aβ_25-35 + anti-miR-NC group. Dual-luciferase reporter gene assay validated the predicted miR-200a-3p binding sites in the 3′-UTR of SIRT1 mRNA. In addition, downregulation of SIRT1 promoted Aβ_25-35-induced neuronal apoptosis and cleaved-caspase-3 level in PC12 cells, whereas anti-miR-200a-3p reversed these effects. Knockdown of SIRT1 decreased the inhibitory effect of Aβ_25-35 on cell viability, while anti-miR-200a-3p attenuated this effect. Overall, the results suggest that suppression of miR-200a-3p attenuates Aβ_25-35-induced apoptosis in PC12 cells by targeting SIRT1. Thus, miR-200a-3p may be a potential therapeutic target for treatment of AD.     

Effects of NMDAR Antagonist on the Regulation of P-MARCKS Protein to Aβ<Subscript>1−42</Subscript> Oligomers Induced Neurotoxicity

Alzheimer’s disease (AD) is a well-known neurodegenerative disease. Deposition of β-amyloid protein (Aβ) oligomers plays a crucial role in the disease progression. Previous studies showed that toxicity induced by Aβ oligomers in cultured neurons and adult rat brain was partially mediated by activation of glutamatergic N-methyl-d-aspartate receptors (NMDAR). Additionally, memantine, a noncompetitive NMDAR antagonist, can significantly improve cognitive functions in some AD patients. However, little is currently known about the potential role of NMDAR antagonist on the regulation of P-MARCKS protein to Aβ_1?42 oligomers induced neurotoxicity. The protective effect and mechanism of NMDAR antagonist on primary neurons exposed to Aβ_1?42 oligomers were investigated in the study. We have defined that the Aβ_1?42 treatment decreased cell viability and increased apoptosis. Moreover, Aβ_1?42 oligomers exposure increased P-MARCKS and PIP2 expressions, while decreased SYP expression. However, NMDAR antagonist pretreatment ameliorates Aβ_1?42 oligomers induced neuronal apoptosis and partially reverses the expression of P-MARCKS, PIP2 and SYP. In conclusion, NMDAR antagonist may ameliorate neurotoxicity induced by Aβ_1?42 oligomers through reducing neuronal apoptosis and protecting synaptic plasticity in rat primary neurons. The mechanism involved may be mediated by the variation of protein P-MARCKS.     

NAD attenuates oxidative DNA damages induced by amyloid beta-peptide in primary rat cortical neurons

Abstract

One major pathological hallmark of Alzheimer's disease (AD) is accumulation of senile plaques in patients’ brains, mainly composed of amyloid beta-peptide (Aβ). Nicotinamide adenine dinucleotide (NAD) has emerged as a common mediator regulating energy metabolism, mitochondrial function, aging, and cell death, all of which are critically involved in neuronal demise observed in AD. In this work, we tested the hypothesis that NAD may attenuate Aβ-induced DNA damages, thereby conferring neuronal resistance to primary rat cortical cultures. We found that co-incubation of NAD dose-dependently attenuated neurotoxicity mediated by Aβ25–35 and Aβ1-42 in cultured rat cortical neurons, with the optimal protective dosage at 50 mM. NAD also abolished the formation of reactive oxygen species (ROS) induced by Aβ25-35. Furthermore, Aβs were capable of inducing oxidative DNA damages by increasing the extents of 8-hydroxy-2´-deoxyguanosine (8-OH-dG), numbers of apurinic/apyrimidinic (AP) sites, genomic DNA single-stranded breaks (SSBs), as well as DNA double-stranded breaks (DSBs)/fragmentation, which can all be attenuated upon co-incubation with NAD. Our results thus reveal a novel finding that NAD is protective against DNA damage induced by existing Aβ, leading ultimately to neuroprotection in primary cortical culture.     

Docosahexaenoic acid supplementation of primary rat hippocampal neurons attenuates the neurotoxicity induced by aggregated amyloid beta protein42 and up-regulates cytoskeletal protein expression

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by extracellular deposits of fibrillar aggregates of amyloid-β peptide (Aβ). Levels of docosahexaenoic acid (DHA, 22:6n-3), the major fatty acid component of the neuronal membrane, are reduced in the AD hippocampus. We hypothesized that hippocampal neurons with reduced DHA levels would be more susceptible to aggregated Aβ-induced death and that this might be overcome by increasing hippocampal neuronal DHA levels. Embryonic Day 18 rat hippocampal cells were cultured in neurobasal medium with B27 supplemented with 0–100 μM DHA for 8 days, then were treated with 5 μM aggregated Aβ₄₂ for 1 day. We found that supplementation with 5–10 μM DHA, which resulted in hippocampal neuron DHA levels of 12–16% of total fatty acids, was optimal for primary hippocampal neuronal survival, whereas supplementation with 5 or 25 μM DHA attenuated aggregated Aβ₄₂-induced neurotoxicity and protected hippocampal neurons, with 25 μM DHA being more effective. DHA supplementation also resulted in significant up-regulation of expression of tyrosine tubulin and acetylated tubulin. We suggest that hippocampal neuronal DHA levels may be critical for AD prevention by attenuating the neurotoxicity induced by Aβ and in maintaining hippocampal neuron survival.