Effects Of Beta-amyloid Peptide Research Articles

Abelson Kinases Mediate the Depression of Spontaneous Synaptic Activity Induced by Amyloid Beta 1-42 Peptides.

Amyloid beta (Aβ) peptides represent one of the most studied etiological factors of Alzheimer's disease. Nevertheless, the effects elicited by different molecular forms of amyloid beta peptides widely vary between the studies, mostly depending on experimental conditions. Despite the enormous amount of accumulated evidences concerning the pathological effects of amyloid beta peptides, the exact identity of the amyloid beta species is still controversial, and even less is clear as regards to the downstream effectors that mediate the devastating impact of these peptides on synapses in the central nervous system. Recent publications indicate that some of the neurotoxic effects of amyloid beta peptides may be mediated via the activation of proteins belonging to the Abelson non-receptor tyrosine kinase (Abl) family, that are known to regulate actin cytoskeleton structure as well as phosphorylate microtubule-associated tau protein, a hallmark of Alzheimer's disease. By performing series of miniature excitatory postsynaptic currents (mEPSC) recordings in cultured hippocampal cells, we demonstrate that activation of Abl kinases by acute application of 42 amino acid-length monomeric amyloid beta (Aβ1-42) peptides reduces spontaneous synaptic release, while this effect can be rescued by pharmacologic inhibition of Abl kinase activity, or by reduction of Abl expression with small interfering RNAs. Our electrophysiological data are further reinforced by a subsequent biochemical analysis, showing enhanced phosphorylation of Abl kinase substrate CT10 Regulator of Kinase-homolog-Like (Crkl) upon treatment of hippocampal neurons with Aβ peptides. Thus, we conclude that Abl kinase activation may be involved in Aβ-induced weakening of synaptic transmission.

Suppressive Effects of Beta Amyloid Peptides (1-42) and (25-35) on Kv1.1 Channel Activity

Beta amyloid (Aβ) peptides have long been implicated in Alzheimer's Disease (AD) pathology, linking them to disruption of: Ca2+ homeostasis, synaptic communication, and long-term potentiation (LTP). But the mechanism(s) underlying these effects are still unclear. Because Kv1.1 and related channels are activated during an action potential, regulate depolarization-produced Ca2+ influx, and inhibition of Kv1 channels can be neurotoxic, we speculate that Aβ-suppression of Kv1 channels may be an early event in AD pathogenesis. Using murine Kv1.1 channels expressed in Xenopus oocytes, we have observed the effects of Aβ(1-42) and the “core” peptide (25-35) on both macro- and micro-scopic currents. Both the bath application of Aβ(1-42) and Aβ(25-35) produced 40-50% suppression of macroscopic Kv1.1 current within 30 m. Suppression of Kv1.1 by Aβ(1-42) was partially dependent on intracellular Ca2+ and PP2B, showing only ∼25% reduction when cells were loaded with BAPTA-AM or exposed to the PP2B-inhibitor cyclosporine A (CsA). Patch-clamp results suggested that Aβ-suppression of Kv1.1 involved both PP2B-dephosphorylation and direct protein-protein interaction of Aβ with Kv1.1. Exposure of the intracellular face of Kv1.1 channels in ripped-off oocyte patches to either catalytically-active PP2B, or Aβ(1-42), produced gradual reductions in p(open), followed by the abrupt disappearance of Kv1.1 activity. Using “tip-dip” artificial membrane methods, Aβ(25-35) exposure eliminated Kv1.1 channel activity when applied to the channel intracellular face. Kv1.1 channels have also been incorporated into Black Lipid Membranes (BLMs), and we are currently assessing the effects of exposure of Aβ to both intra- and extra-cellular faces of Kv1.1. Suppression of presynaptic Kv1.1 (and related) channels could lead to enhanced action potentials, thus allowing greater influx of Ca2+ and subsequent increase in glutamate release. Postsynaptically, the increased glutamate release, through activation of AMPA and NMDA receptors, may contribute to excitotoxicity.

The Effect of Beta-Amyloid Peptides and Main Stress Protein HSP70 on Human SH-SY5Y Neuroblastoma Proteome

The Effect of Beta-Amyloid Peptides and Main Stress Protein HSP70 on Human SH-SY5Y Neuroblastoma Proteome

Cannabidiol Reverses Deficits in Hippocampal LTP in a Model of Alzheimer's Disease.

Here we demonstrate for the first time that cannabidiol (CBD) acts to protect synaptic plasticity in an in vitro model of Alzheimer's disease (AD). The non-psycho active component of Cannabis sativa, CBD has previously been shown to protect against the neurotoxic effects of beta amyloid peptide (Aβ) in cell culture and cognitive behavioural models of neurodegeneration. Hippocampal long-term potentiation (LTP) is an activity dependent increase in synaptic efficacy often used to study cellular mechanisms related to memory. Here we show that acute application of soluble oligomeric beta amyloid peptide (Aβ1-42) associated with AD, attenuates LTP in the CA1 region of hippocampal slices from C57Bl/6 mice. Application of CBD alone did not alter LTP, however pre-treatment of slices with CBD rescued the Aβ1-42 mediated deficit in LTP. We found that the neuroprotective effects of CBD were not reversed by WAY100635, ZM241385 or AM251, demonstrating a lack of involvement of 5HT1A, adenosine (A2A) or Cannabinoid type 1 (CB1) receptors respectively. However in the presence of the PPARγ antagonist GW9662 the neuroprotective effect of CBD was prevented. Our data suggests that this major component of Cannabis sativa, which lacks psychoactivity may have therapeutic potential for the treatment of AD.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype.

Aβ42 oligomers impair the bioenergetic activity in hippocampal synaptosomes derived from APP-KO mice.

Employing hippocampal synaptosomes from amyloid precursor protein (APP)-deleted mice we analyzed the immediate effects of amyloid beta peptide 42 (Aβ42) peptide in its oligomeric or fibrillar assembly or of soluble amyloid precursor protein alpha (sAPPα) protein on their bioenergetic activity. Upon administration of oligomeric Aβ42 peptide for 30 min we observed a robust decrease both in mitochondrial activity and in mitochondrial membrane potential (MMP). In contrast the respective fibrillary or scrambled peptides showed no effect, indicating that inhibition strictly depends on the oligomerization status of the peptide. Hippocampal synaptosomes from old APP-KO mice revealed a further reduction of their already impaired bioenergetic activity upon incubation with 10 μm Aβ42 peptide. In addition we evaluated the influence of the sAPPα protein on mitochondrial activity of hippocampal synaptosomes derived from young or old APP-KO animals. In neither case 20 nm nor 200 nm sAPPα protein had an effect on mitochondrial metabolic activity. Our findings demonstrate that hippocampal synaptosomes derived from APP-KO mice are a most suitable model system to evaluate the impact of Aβ42 peptide on its bioenergetic activity and to further elucidate the molecular mechanisms underlying the impairments by oligomeric Aβ42 on mitochondrial function. Our data demonstrate that extracellular Aβ42 peptide is taken up into synaptosomes where it immediately attenuates mitochondrial activity.

The Effects of Beta Amyloid Peptide 1-42 on Isolated Rat Hearts and Ileum Smooth Muscle

Neurotoxic beta amyloid peptides (βAPs) are involved in the pathogenesis of Alzheimer disease. βAP_1-42 may also play a role in the regulation of cardiovascular functions. Therefore, we investigated the possible effects of βAP_1-42 on isolated rat heart and ileum. The hearts were perfused with modified Krebs-Henseleit solution. Left ventricular developed pressure (LVDP), maximal rate of pressure development of left ventricle (+dP/dt_max), heart rate, coronary flow, monophasic action potential amplitude (MAPamp), MAP duration at 90% repolarization (MAP₉₀) and contractions of ileum were measured. One, 10 and 100 nmol/l doses of βAP_1-42 significantly decreased LVDP, +dP/dt_max and heart rate. The dose of 1 nmol/l did not change coronary flow, but 10 and 100 nmol/l doses significantly reduced it. All doses of βAP_1-42 did not alter MAPamp, but increased MAP₉₀. βAP_1-42 (1, 10, 100, 1,000 nmol/l) also did not influence ileum contractions. We suggest that βAP_1-42 produces negative inotropic and negative chronotropic effects with an increase in MAP duration. Furthermore, βAP_1-42 at high doses decreases coronary flow.

Amyloid Beta Peptides Affect Pregnenolone and Pregnenolone Sulfate Levels in PC-12 and SH-SY5Y Cells Depending on Cholesterol.

Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25-35, AB 1-40 and AB 1-42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose-and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25-35 were higher than those in AB 1-40 and AB 1-42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase might be playing a key role in the cell death mechanism of AB toxicity depending on cellular cholesterol levels.

Toxicity of amyloid beta 1-40 and 1-42 on SH-SY5Y cell line

Objectives: Amyloid beta plaques are primary hallmark of Alzheimer’s disease, which is characterized by specific neurodegeneration. Amyloid beta peptide – the main plaque component- was shown to be neurotoxic in animal models, primary neuronal cultures and immortalized cell lines. However, the results are often controversial and there is no good human cell line model for evaluation of the toxicity of amyloid peptides. Here we studied the effect of amyloid beta 1-40 and 1-42 on undifferentiated and differentiated human neuroblastoma cell line SH-SY5Y. Results: Undifferentiated cell culture was too diverse and unstable to reveal a toxic effect of amyloid beta peptides quantitatively. Differentiated cells established more neuron-like phenotype and were more identical and stable in culture suggesting potential susceptibility to amyloid beta as a neurotoxic agent. Amyloid peptides are prone to form different aggregates with diverse toxic properties, in current study, monomeric amyloid beta 1-40 and 1-42 were applied to the cells. Viability test WST-1 and propidium iodide (PI) uptake tests showed that undifferentiated cells are not susceptible to amyloid beta, however, differentiated cells showed reduced viability and increased PI uptake in case of amyloid beta 1-42, but not in case of amyloid beta 1-40. Conclusions: Current study revealed that amyloid beta has no remarkably toxic effect on undifferentiated SH-SY5Y cell line whereas viability of the neuron-like differentiated cell culture is significantly decreased by the amyloid beta 1-42 peptide that is known to form spontaneously toxic aggregates.

Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide (25-35).

In this study, we treated PC12 cells with 0–20 μM amyloid-β peptide (25–35) for 24 hours to induce cytotoxicity, and found that 5–20 μM amyloid-β peptide (25–35) decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease in PC12 cell viability induced by amyloid-β peptide (25–35). Diazoxide protected PC12 cells against amyloid-β peptide (25–35)-induced increases in mitochondrial membrane potential and intracellular reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nω-nitro-L-arginine, also protected PC12 cells from amyloid-β peptide (25–35)-induced increases in both mitochondrial membrane potential and intracellular reactive oxygen species levels. However, the H2O2-degrading enzyme catalase could not reverse the amyloid-β peptide (25–35)-induced increase in intracellular reactive oxygen species. A 24-hour exposure to amyloid-β peptide (25–35) did not result in apoptosis or necrosis, suggesting that the increases in both mitochondrial membrane potential and reactive oxygen species levels preceded cell death. The data suggest that amyloid-β peptide (25–35) cytotoxicity is associated with adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β peptide (25–35).

Effects of Abeta1–42 on the current of KATP channels in cultured cholinergic neurons

Objective: This study mainly determined the effect of beta-amyloid peptides (Abeta1–42) on the ATP-sensitive potassium (KATP) channels of the cultured cortical and hippocampal cholinergic neurons, and further confirmed the protective effect of Diazoxide, which was the specific opener of KATP channels, to the neurons.Methods: The primary rat cortical and hippocampal cholinergic neurons were cultured. Membrane currents were measured using whole-cell patch-clamp technique.Results: The data showed that the outward current of neurons significantly decreased after using Abeta1–42 (P<0·05), and that the outward current of decreased did not change after given Diazoxide again (P>0·05) compared with control. However, after being pretreated with Diazoxide for 1 hour before using Abeta1–42, this phenomenon did not exist; that is, the membrane current of the neurons did not change when we give Abeta1–42 after pretreatment with Diazoxide (P>0·05).Conclusions: It is concluded that Abeta1–42 inhibits the openings of the KATP channels, while this inhibitory action can be removed by pretreatment with Diazoxide. It is indicated that the opening of KATP channels may play a potential neuroprotective role in antineurotoxicity of Abeta1–42, and the application of Diazoxide in small dose may be helpful in the treatment of Abeta1–42 neurotoxicity.

Hypoxia enhances stimulating effect of amyloid beta peptide (25–35) for interleukin 17 and T helper lymphocyte subtype 17 upregulation in cultured peripheral blood mononuclear cells

Th17 has been demonstrated to have a key role in several autoimmune diseases. The present study was carried out to investigate changes in IL-17 and Th17 in cultured PBMC in response to Abeta peptide (25-35) and hypoxic stimulation in vitro. PBMC were collected from adult healthy donors and cultured in normal and anaerobic conditions (hypoxia 1, 3, 6, 12, 24 hr). Each group of cells was stimulated with Abeta peptide (25-35; 3, 10 nmol/ml). ELISA was used to examine IL-17A concentrations in the supernatants, and flow cytometry for the numbers of IL-17A secreting CD4 positive Th17 lymphocytes. Statistically significant increases in IL-17A and Th17 concentrations were found in groups with 10 nmol/ml Abeta (25-35) and more than three hr anaerobic culture. IL-17A and Th17 concentrations in anaerobic groups increased gradually with time and peaked at six hr. Compared with other groups, the highest concentrations were found in those treated with 10 nmol/ml Abeta and cultured for six hr (P < 0.001). This study provides the first report that IL-17A and Th17 lymphocytes are possibly involved in the immune pathogenesis caused by Abeta peptide (25-35). Hypoxia may enhance this response independently of time.

Differential effects of interleukin-1&amp;beta; and S100B on amyloid precursor protein in rat retinal neurons

Interleukin-1beta (IL-1beta) and S100B calcium binding protein B (S100B) have been implicated in the pathogenesis of Alzheimer's disease. Both are present in and around senile plaques and have been shown to increase levels of amyloid precursor protein (APP) mRNA in vitro. However, it is not known how either of these substances affects APP in vivo. We have studied the effects of IL-1beta and S100B on the expression and processing of APP using a retinal-vitreal model. We have also investigated the effect of amyloid beta peptide (Abeta) on APP in the same system and the regulation of S100B production by Abeta and IL-1beta from retinal glial cells. Retinal ganglion cells constitutively express APP. However, after intravitreal injection of IL-1beta or Abeta there was a marked reduction in APP levels as detected by Western blotting and IL-1beta produced a decrease in APP immunoreactivity (IR). Nissl staining showed that the integrity of the injected retinas was unchanged after injection. Two days after S100B injection, there was a small reduction in APP-IR but this was accompanied by the appearance of some intensely stained large ganglion cells and there was some up-regulation in APP holoprotein levels on Western blot. Seven days post-S100B injection, these large, highly stained cells had increased in number throughout the retina. Injection of Abeta and IL-1beta also caused an increase in S100B production within the retinal Müller glial cells. These results support the hypothesis that S100B (a glial-derived neurotrophic factor) and IL-1beta (a pro-inflammatory cytokine) can modulate the expression and processing of APP in vivo and so may contribute to the progression of Alzheimer's disease.

Up-regulation of astrocyte metabotropic glutamate receptor 5 by amyloid-β peptide

The effects of amyloid-beta peptide (Aβ) on astrocyte responses to activation of mGlu5 receptors have been investigated using calcium imaging. Pre-incubation with Aβ1–40 peptide for up to 72 h produced a time- and concentration-dependent 2–4 fold enhancement in the magnitude of the intracellular calcium mobilization response to the group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG). In contrast, pre-treatment with Aβ1–40 did not alter the calcium responses induced by other G protein coupled- or ion channel-receptors. Aβ 1–40-enhanced DHPG responses were blocked by the mGlu5 antagonist MPEP but not by inhibitors of voltage dependent calcium channels or by the AMPA/KA receptor antagonist CNQX. Up-regulation of mGlu5 coupled responses was associated with significant increases in astrocyte mGlu5 receptor- mRNA and -protein expression after preincubation with Aβ . The changes observed in vitro were consistent with results obtained from human Alzheimer's disease (AD) patients. Immunostaining for mGlu5 receptors was increased on astrocytes which were colocalized with Aβ plaques in hippocampal tissue from AD patients compared to age-matched controls. These results suggest that modulation of mGlu5 receptors in astrocytes could be an important mechanism in determining the progression of pathology in AD.

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo

Amyloid-β (Aβ) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Aβ peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Aβ25–35 and Aβ1–40 for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Aβ1–40 led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Aβ peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.

Complex of Amyloid β Peptides with 24-Hydroxycholesterol and Its Effect on Hemicholinium-3 Sensitive Carriers

Brains of Alzheimer disease patients in early stages of dementia contain an increased 24(S)-hydroxycholesterol (cerebrosterol)/cholesterol ratio when compared to controls. In this study, effects of amyloid beta peptides and of racemic 24-hydroxycholesterol were evaluated in vitro on undepleted or cholesterol-depleted hippocampal synaptosomes of young and old rats via a high-affinity choline transport and membrane anisotropy measurements. Depletion of membrane cholesterol decreased the transport of [3H]choline, increased the specific binding of [3H]hemicholinium-3 and decreased membrane anisotropy. However, less alterations were found in old when compared to young brains. 500 nM nonaggregated peptides were ineffective but aggregated fragment 1-42 evoked marked drops in the transport and anisotropy values on depleted synaptosomes. 50 microM 24-hydroxycholesterol inhibited choline transport on depleted synaptosomes but it did not influence membrane anisotropy. Peptides eliminated the actions of oxysterol on choline carriers in young but not in old rats. On the other hand, oxysterol eliminated the effects of peptides on membrane anisotropy. Our study suggests a possible role of membrane cholesterol in the regulation of choline carriers and supports data reporting a protective role of membrane cholesterol against toxic effects of amyloid beta peptides. Moreover, via Raman spectroscopy we demonstrate for the first time that peptides form a complex with 24-hydroxycholesterol.

Chronic but not acute intracerebroventricular administration of amyloid β‐peptide(25–35) decreases somatostatin content, adenylate cyclase activity, somatostatin‐induced inhibition of adenylate cyclase activity, and adenylate cyclase I levels in the rat hippocampus

Although alterations in adenylate cyclase (AC) activity and somatostatin (SRIF) receptor density have been reported in Alzheimer's disease, the effects of amyloid beta-peptide (Abeta) on these parameters in the hippocampus are unknown. Our aim was to investigate whether the peptide fragment Abeta(25-35) can affect the somatostatinergic system in the rat hippocampus. Hence, Abeta(25-35) was injected intracerebroventricularly (i.c.v.) to Wistar rats in a single dose or infused via an osmotic minipump connected to a cannula implanted in the right lateral ventricle during 14 days. The animals were decapitated 7 or 14 days after the single injection and 14 days after chronic infusion of the peptide. Chronic i.c.v. infusion of Abeta(25-35) decreased SRIF-like immunoreactive content without modifying the SRIF receptor density, SRIF receptor expression, or the Gialpha(1), Gialpha(2), and Gialpha(3) protein levels in the hippocampus. This treatment, however, caused a decrease in basal and forskolin-stimulated AC activity as well as in the capacity of SRIF to inhibit AC activity. Furthermore, the protein levels of the neural-specific AC type I were significantly decreased in the hippocampus of the treated rats, whereas an increase in the levels of AC V/VI was found, with no alterations in type VIII AC. A single i.c.v. dose of Abeta(25-35) exerted no effect on SRIF content or SRIF receptors but induced a slight decrease in forskolin-stimulated AC activity and its inhibition by SRIF. Because chronic Abeta(25-35) infusion impairs learning and memory whereas SRIF facilitates these functions, the alterations described here might be physiologically important given the decreased cognitive behavior previously reported in Abeta-treated rats.

Influence of beta-amyloid peptide on cell membrane lipids and cholinergic receptors in human neuroblastoma SH-SY5Y cells

To study the effects of beta-amyloid peptide (Abeta) on cell membrane lipids and cholinergic receptors of human neuroblastoma cells. Human SH-SY5Y neuroblastoma cells were treated with different concentrations of Abeta(1-42) with and without pretreatment of vitamin E. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction, lipid peroxidation, protein oxidation and phospholipids were measured by spectrophotometry. Levels of cholesterol and unbiquinone were determined by high-performance liquid chromatography (HPLC). The numbers of cholinergic receptor binding sites were determined by receptor binding assay and the protein levels of nicotinic receptor alpha3 and alpha7 subunits were studied by Western blotting. SH-SY5Y cells showed decreased reduction rates of MMT and phospholipids, and increased lipid peroxidation and protein oxidation after exposure to Abeta (0.1 micromol/L) as compared to the control. The number of cholinergic receptor binding sites, the protein level of nicotinic receptor alpha3 and alpha7 subunits and the content of ubiquinone decreased in cells treated with high dose of Abeta (1 micromol/L). Although the level of cholesterol was not changed in any way, vitamin E partially prevented the neurotoxic effects of Abeta. beta-amyloid peptide reduces the level of cell membrane lipids and cholinergic receptors in human SH-SY5Y neuroblastoma cells, likely through the induction of an enhanced oxidative stress.

Quantitative Proteomic Analysis of Mitochondria from Primary Neuron Cultures Treated with Amyloid Beta Peptide

Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of neuron degeneration in Alzheimer's disease (AD). Although several studies have examined the effect of amyloid beta peptide (Abeta), on activities of individual proteins in primary neuron cultures, there have been no studies of the effects of Abeta on the mitochondrial proteome. Here, we quantitatively measured changes in mitochondrial proteins of primary rat cortical neuron cultures exposed to 25 microM Abeta(25-35) for 16 h using isotope coded affinity tag (ICAT) labeling and 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) which allows simultaneous identification and quantification of cysteine-containing proteins. The analysis of enriched mitochondrial fractions identified 10 proteins including sodium/potassium-transporting ATPase, cofilin, dihydropyrimidinase, pyruvate kinase and voltage dependent anion channel 1 that were statistically significantly (P < 0.05) altered in Abeta-treated cultures. Elevations of proteins associated with energy production suggest that cells undergoing Abeta-mediated apoptosis increase synthesis of proteins essential for ATP production and efflux in an attempt to maintain metabolic function.