Acetyl-L-Carnitine Arginine Amide Prevents β 25–35-Induced Neurotoxicity in Cerebellar Granule Cells

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with 25–35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KC1 in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later -amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with 25–35 was able to rescue neurons from 25–35-induced neurotoxicity. We also studied the changes in Ca²⁺ homeostasis following glutamate stimulation, in control and -amyloid treated single cells, either in presence or in absence of ST857. 25–35 did not affect basal [Ca²⁺]_i, while modified glutamate-induced [Ca²⁺]_i increase, causing a sustained plateau phase of [Ca²⁺]_i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with 25–35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca²⁺ homeostasis.     

Selenium Compounds Prevent Amyloid β-Peptide Neurotoxicity in Rat Primary Hippocampal Neurons

Neuropathological hallmarks of Alzheimer’s disease (AD) include amyloid plaque formation, neurofibrillary tangles, neuronal and synaptic loss. This study aims to identify the neuroprotective effects of the selenium compounds on the neurotoxicity of amyloid β(1–42) in primary cultures of murine hippocampal neurons. Samples were subjected to immunocytochemistry and western blotting techniques to determine the role of treatments on neuronal viability and synaptic protein SNAP-25. We observed a reduced cell viability amyloid β-peptide (1–42)-induced. When cells were co-treated with amyloid β-peptide (1–42) and selenium compounds, we verified a strong increase in relative cell viability and in the level of synaptic marker synaptosomal-associated protein SNAP-25 induced by selenium compounds.     

Effects of Modeling of Hypercalcemia and β-Amyloid on Cultured Hippocampal Neurons of Rats

Neurophysiology - Alzheimer’s disease (AD) is the most common type of dementia; it is characterized by accumulations of amyloid (Aβ) plaques and neurofibrillary tangles in the brain....     

The Ketone Body, β-Hydroxybutyrate Stimulates the Autophagic Flux and Prevents Neuronal Death Induced by Glucose Deprivation in Cortical Cultured Neurons

Glucose is the major energy substrate in brain, however, during ketogenesis induced by starvation or prolonged hypoglycemia, the ketone bodies (KB), acetoacetate and β-hydroxybutyrate (BHB) can substitute for glucose. KB improve neuronal survival in diverse injury models, but the mechanisms by which KB prevent neuronal damage are still not well understood. In the present study we have investigated whether protection by the D isomer of BHB (D-BHB) against neuronal death induced by glucose deprivation (GD), is related to autophagy. Autophagy is a lysosomal-dependent degradation process activated during nutritional stress, which leads to the digestion of damaged proteins and organelles providing energy for cell survival. Results show that autophagy is activated in cortical cultured neurons during GD, as indicated by the increase in the levels of the lipidated form of the microtubule associated protein light chain 3 (LC3-II), and the number of autophagic vesicles. At early phases of glucose reintroduction (GR), the levels of p62 declined suggesting that the degradation of the autophagolysosomal content takes place at this time. In cultures exposed to GD and GR in the presence of D-BHB, the levels of LC3-II and p62 rapidly declined and remained low during GR, suggesting that the KB stimulates the autophagic flux preventing autophagosome accumulation and improving neuronal survival.     

Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.     

β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons

β-Amyloid (Aβ) peptides may cause malfunction and death of neurons in Alzheimer’s disease. We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Aβ(25-35) at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal–glial cell cultures (from P1 rats), Aβ reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Aβ(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Aβ may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake. Special issue article in honor of Dr. Frode Fonnum.     

Intracellular Ion Channel CLIC1: Involvement in Microglia-Mediated β-Amyloid Peptide(1-42) Neurotoxicity

Microglia can exacerbate central nervous system disorders, including stroke and chronic progressive neurodegenerative diseases such as Alzheimer disease. Mounting evidence points to ion channels expressed by microglia as contributing to these neuropathologies. The Chloride Intracellular Channel (CLIC) family represents a class of chloride intracellular channel proteins, most of which are localized to intracellular membranes. CLICs are unusual in that they possess both soluble and integral membrane forms. Amyloid β-peptide (Aβ) accumulation in plaques is a hallmark of familial Alzheimer disease. The truncated Aβ_25-35 species was shown previously to increase the expression of CLIC1 chloride conductance in cortical microglia and to provoke microglial neurotoxicity. However, the highly pathogenic and fibrillogenic full-length Aβ_1-42 species was not examined, nor was the potential role of CLIC1 in mediating microglial activation and neurotoxicity by other stimuli (e.g. ligands for the Toll-like receptors). In the present study, we utilized a two chamber Transwell? cell culture system to allow separate treatment of microglia and neurons while examining the effect of pharmacological blockade of CLIC1 in protecting cortical neurons from toxicity caused by Aβ_1-42- and lipopolysaccaride-stimulated microglia. Presentation of Aβ_1-42 to the upper, microglia-containing chamber resulted in a progressive loss of neurons over 3 days. Neuronal cell injury was prevented by the CLIC1 ion channel blockers IAA-94 [(R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] and niflumic acid (2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid) when presented to the upper chamber only. Incubation of microglia with lipopolysaccharide plus interferon-γ led to neuronal cell injury which, however, was insensitive to inhibition by the CLIC1 channel blockers, suggesting a degree of selectivity in agents leading to CLIC1 activation.     

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.     

Cryptotanshinione Inhibits β-Amyloid Aggregation and Protects Damage from β-Amyloid in SH-SY5Y Cells

The deposition of amyloid β-protein (Aβ) fibrils into plaques within the brain parenchyma and along cerebral blood vessels is a hallmark of Alzheimer’s disease (AD). Aβ42 oligomers and fibrils cause the breakdown of neural circuits, neuronal death and eventually dementia. Drugs that inhibit Aβ42 aggregation may be a novel direction in AD drug discovery. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of AD. However, the effects of CTS on the Aβ aggregation and toxicity are unclear. The current work shows the effectiveness of CTS on the inhibition of Aβ42 aggregation and toxicity to human neuroblastoma cells. In this study, we demonstrated that CTS can inhibit Aβ42 spontaneous aggregation using thioflavin T fluorescence assay and transmission electron microscopy. Furthermore, we investigated the effects of CTS on Aβ-induced oxidative cell death in cultured SH-SY5Y cells. MTT and lactate dehydrogenase assays showed that CTS reduced the cytotoxicity induced by Aβ42. CTS also dramatically reduced Aβ42-induced cellular apoptosis and increased level of reactive oxygen species in these cells. Our study suggests that CTS may be useful in the inhibition or prevention of AD development and progression.     

αν and β1 Integrins Mediate Aβ-Induced Neurotoxicity in Hippocampal Neurons via the FAK Signaling Pathway

αν and β1 integrins mediate Aβ–induced neurotoxicity in primary hippocampal neurons. We treated hippocampal neurons with 2.5 µg/mL 17E6 and 5 µg/mL ab58524, which are specific αν and β1 integrin antagonists, respectively, for 42 h prior to 10 µM Aβ treatment. Next, we employed small interfering RNA (siRNA) to silence focal adhesion kinase (FAK), a downstream target gene of integrins. The siRNAs were designed with a target sequence, an MOI of 10 and the addition of 5 µg/mL polybrene. Under these conditions, the neurons were transfected and the apoptosis of different cell types was detected. Moreover, we used real-time PCR and Western blotting analyses to detect the expression of FAK and ρFAK genes in different cell types and investigated the underlying mechanism and signal pathway by which αν and β1 integrins mediate Aβ-induced neurotoxicity in hippocampal neurons. An MTT assay showed that both 17E6 and ab58524 significantly increased cell viability compared with the Aβ-treated neurons (P<0.01 and P<0.05, respectively). However, this protective effect was markedly attenuated after transfection with silencing FAK (siFAK). Moreover, TUNEL immunostaining and flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ (P<0.05) compared with the Aβ-treated cells. However, this protective effect was reversed with siFAK treatment. Both the gene and protein expression of FAK increased after Aβ treatment. Interestingly, as the gene and protein levels of FAK decreased, the ρFAK protein expression markedly increased. Furthermore, both the gene and protein expression of FAK and ρFAK were significantly diminished. Thus, we concluded that both αν and β1 integrins interfered with Aβ-induced neurotoxicity in hippocampal neurons and that this mechanism partially contributes to the activation of the Integrin-FAK signaling pathway.     

Effects of Aβ1–42 on the Subunits of KATP Expression in Cultured Primary Rat Basal Forebrain Neurons

ATP-sensitive potassium channels (KATP) play a crucial role in coupling metabolic energy to the membrane potential of cells, thereby functioning as cellular "metabolic sensors." Recent evidence has showed a connection between the amyloid neurotoxic cascade and metabolic impairment. With regard to their neuroprotection in other neuronal preparations, KATP channels may mediate a potential neuroprotective role in Alzheimer's disease (AD). To investigate the effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain cholinergic neurons, primary rat basal forebrain neurons were cultured and evaluated. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing changes were observed by double immunofluorescence and immunoblotting when the neurons were exposed to Abeta1-42(2 microM) for different time (0, 24, 72 h). We found a significant increase in the expression of Kir6.1 and SUR2 in the cultured neurons being exposed to Abeta1-42 for 24 h, while Kir6.2 and SUR1 showed no significant change. However, after being treated with Abeta1-42 for 72 h, the expression of the four subunits was all increased significantly compared with the control. These findings suggest that being exposed to Abeta1-42 for different time (24 and 72 h) induces differential regulations of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to resist the toxicity of Abeta1-42.     

Geniposide Protects Primary Cortical Neurons against Oligomeric Aβ1-42-Induced Neurotoxicity through a Mitochondrial Pathway

Mitochondrial dysfunction plays a key role in the progression of Alzheimer’s disease (AD). The accumulation of amyloid-beta peptide (Aβ) in the brains of AD patients is thought to be closely related to neuronal mitochondrial dysfunction and oxidative stress. Therefore, protecting mitochondria from Aβ-induced neurotoxicity is an effective strategy for AD therapeutics. In a previous study, we found that geniposide, a pharmacologically active compound purified from gardenia fruit, has protective effects on oxidative stress and mitochondrial dysfunction in AD transgenic mouse models. However, whether geniposide has a protective effect on Aβ-induced neuronal dysfunction remains unknown. In the present study, we demonstrate that geniposide protects cultured primary cortical neurons from Aβ-mediated mitochondrial dysfunction by recovering ATP generation, mitochondrial membrane potential (MMP), and cytochrome c oxidase (CcO) and caspase 3/9 activity; by reducing ROS production and cytochrome c leakage; as well as by inhibiting apoptosis. These findings suggest that geniposide may attenuate Aβ-induced neuronal injury by inhibiting mitochondrial dysfunction and oxidative stress.     

Kukoamine A Protects against NMDA-Induced Neurotoxicity Accompanied with Down-Regulation of GluN2B-Containing NMDA Receptors and Phosphorylation of PI3K/Akt/GSK-3β Signaling Pathway in Cultured Primary Cortical Neurons

Neurochemical Research - Kukoamine (KuA) is a spermine alkaloid present in traditional Chinese medicine Cortex Lycii radices, which possesses various pharmacological properties. Our previous...     

BDNF-, IGF-1- and GDNF-Secreting Human Neural Progenitor Cells Rescue Amyloid β-Induced Toxicity in Cultured Rat Septal Neurons

Alzheimer’s disease (AD) is characterized by the depositions of amyloid-β (Aβ) proteins, resulting in a reduction of choline acetyltransferase (ChAT) activity of AD brain in the early stages of the disease. Several growth factors, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative both in vitro and in vivo models. In this study, septal neurons were prepared from septal nucleus of embryonic (day 16-17) rat brain and treated with monomeric, oligomeric or fibrillar Aβ_1-42 peptide. Oligomeric Aβ_1-42, (10 μM) was the most potent at sublethal dose. Septal neuron cultures treated with BDNF, IGF-1 or GDNF or co-cultured with genetically modified human neural progenitor cells (hNPCs) secreting these neurotrophic factors (but not allowing contact between the two cell types), were protected from oligomeric Aβ_1-42 peptide-induced cell death, and these trophic factors enhanced cholinergic functions by increasing ChAT expression level. These results indicate the potential of employing transplanted hNPCs for treatment of AD.     

Statins Reduce Amyloid β-Peptide Production by Modulating Amyloid Precursor Protein Maturation and Phosphorylation Through a Cholesterol-Independent Mechanism in Cultured Neurons

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2–2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.     

Antigen-Bound and Free β-Amyloid Autoantibodies in Serum of Healthy Adults

Physiological β-amyloid autoantibodies (Aβ-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aβ antigen-antibody immune complexes. Based on the epitope specificity of the Aβ-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aβ-IgG immune complexes and (b) an indirect ELISA for the determination of free Aβ-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aβ-IgG immune complexes compared to free Aβ-autoantibodies, while no correlation with age or cognitive performance of the participants was found.     

Effects of a Standardized Phenolic-Enriched Maple Syrup Extract on β-Amyloid Aggregation,Neuroinflammation in Microglial and Neuronal Cells,and β-Amyloid Induced Neurotoxicity in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Published data supports the neuroprotective effects of several phenolic-containing natural products, including certain fruit, berries, spices, nuts, green tea, and olive oil. However, limited data are available for phenolic-containing plant-derived natural sweeteners including maple syrup. Herein, we investigated the neuroprotective effects of a chemically standardized phenolic-enriched maple syrup extract (MSX) using a combination of biophysical, in vitro, and in vivo studies. Based on biophysical data (Thioflavin T assay, transmission electron microscopy, circular dichroism, dynamic light scattering, and zeta potential), MSX reduced amyloid β_1?42 peptide (Aβ_1?42) fibrillation in a concentration-dependent manner (50–500 μg/mL) with similar effects as the neuroprotective polyphenol, resveratrol, at its highest test concentration (63.5?% at 500 μg/mL vs. 77.3?% at 50 μg/mL, respectively). MSX (100 μg/mL) decreased H₂O₂-induced oxidative stress (16.1?% decrease in ROS levels compared to control), and down-regulated the production of lipopolysaccharide (LPS)-stimulated inflammatory markers (22.1, 19.9, 74.8, and 87.6?% decrease in NOS, IL-6, PGE₂, and TNFα levels, respectively, compared to control) in murine BV-2 microglial cells. Moreover, in a non-contact co-culture cell model, differentiated human SH-SY5Y neuronal cells were exposed to conditioned media from BV-2 cells treated with MSX (100 μg/mL) and LPS or LPS alone. MSX-BV-2 media increased SH-SY5Y cell viability by 13.8?% compared to media collected from LPS-BV-2 treated cells. Also, MSX (10 μg/mL) showed protective effects against Aβ_1?42 induced neurotoxicity and paralysis in Caenorhabditis elegans in vivo. These data support the potential neuroprotective effects of MSX warranting further studies on this natural product.