Androgens regulate neprilysin expression: role in reducing beta-amyloid levels

Age-related testosterone depletion in men is a risk factor for Alzheimer's disease. Prior studies suggest that androgens affect Alzheimer's disease risk by regulating accumulation of β-amyloid protein (Aβ) by an undefined mechanism. In this study, we investigated the role of the Aβ-catabolizing enzyme neprilysin (NEP) in this process. First, we observed that androgens positively regulate neural expression of NEP in adult male rats. Next, we investigated androgen regulatory effects on both NEP expression and Aβ levels using cultured hippocampal neurons and neuronally differentiated rat pheochromocytoma cell 12 with or without androgen receptor (AR). Dihydrotestosterone (DHT) induced a time-dependent increase in NEP expression. DHT also significantly decreased levels of Aβ in AR-expressing cells transfected with amyloid precursor protein, but did not affect levels of either full-length or non-amyloidogenic, soluble amyloid precursor protein. Importantly, the DHT induced decrease of Aβ was blocked by pharmacological inhibition of NEP. The DHT-mediated increase in NEP expression and decrease in Aβ levels were (i) not observed in rat pheochromocytoma cell 12 lacking AR and (ii) blocked in AR-expressing cells by the antagonists, cyproterone acetate and flutamide. Together, these findings suggest that androgen regulation of Aβ involves an AR-dependent mechanism requiring up-regulation of the Aβ catabolizing enzyme NEP.     

Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle

Autosomal recessive hereditary inclusion-body myopathy (h-IBM) is caused by mutations of the UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase gene, a rate-limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h-IBM. h-IBM muscle shows the abnormal accumulation of proteins including amyloid-β (Aβ). Neprilysin (NEP), a metallopeptidase that cleaves Aβ, is characterized by the presence of several N-glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h-IBM muscles analyzed. In vitro , the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post-translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Aβ mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h-IBM muscle, hyposialylated NEP has a role in hampering the cellular Aβ clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo-lytic fragment of the membrane-associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial "plaques" created from aggregated synthetic Aβ peptides. These correspond to full-length Aβ residues 1–40 (Aβ1–40), an internal β-sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ-neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly-peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.     

Amyloid β-Protein Induces Its Own Production in Cultured Degenerating Cerebrovascular Smooth Muscle Cells

Abstract: The progression of Alzheimer's disease and related disorders involves amyloid β-protein (Aβ) deposition and pathologic changes in the parenchyma as well as cerebral blood vessels. The cerebrovascular Aβ deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall, which have been shown to express the Aβ precursor (AβPP) and Aβ. Here, we show that Aβ_1–42, an abundant cerebrovascular form of Aβ, causes cellular degeneration in cultured human cerebrovascular smooth muscle cells. This stress response is accompanied by a striking increase in the levels of cellular AβPP and soluble Aβ peptide produced in these degenerating cells. These data provide the first experimental evidence that Aβ can potentially contribute to the onset and progression of the cerebrovascular pathology. The present findings suggest that this mechanism may involve a molecular cascade with a novel product-precursor relationship that results in the adverse production and subsequent accumulation of Aβ.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

A novel reciprocal and biphasic relationship between membrane cholesterol and β-secretase activity in SH-SY5Y cells and in human platelets

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the Aβ peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane β-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane β-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment ( n  = 172) were significantly more likely to lie within the negative correlation zone than control platelets ( n  = 171). Pharmacological inhibition of SH-SY5Y β-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane β-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

Susceptibility of amyloid beta peptide degrading enzymes to oxidative damage: a potential Alzheimer's disease spiral

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.     

Proinflammatory cytokine interferon-γ increases induction of indoleamine 2,3-dioxygenase in monocytic cells primed with amyloid β peptide 1–42: implications for the pathogenesis of Alzheimer's disease

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5–25 μM amyloid β peptide (Aβ) (1–42) but not with Aβ (1–40) or Aβ (25–35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-γ (IFN-γ) but not with interleukin-1β, tumor necrosis factor-α, or interleukin-6 at 100 U/mL. The concomitant addition of Aβ (1–42) with IFN-γ was totally ineffective, indicating that Aβ pre-treatment is prerequisite for a high IDO expression. The priming effect of Aβ (1–42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-γ induces IDO over-expression in the primed microglia surrounding amyloid plaques.     

Alzheimer's β-amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2

Beta-amyloid peptides (Aβ) are the major constituents of senile plaques and cerebrovascular deposits in the brains of Alzheimer's disease patients. We have shown previously that soluble forms of Aβ are anti-angiogenic both in vitro and in vivo . However, the mechanism of the anti-angiogenic activity of Aβ peptides is unclear. In this study, we examined the effects of Aβ1–42 on vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, which plays a key role in angiogenesis. Aβ inhibited VEGF-induced migration of endothelial cells, as well as VEGF-induced permeability of an in vitro model of the blood brain barrier. Consistently, exogenous VEGF dose-dependently antagonized the anti-angiogenic activity of Aβ in a capillary network assay. Aβ1–42 also blocked VEGF-induced tyrosine phosphorylation of VEGFR-2 in two types of primary endothelial cells, suggesting an antagonistic action of Aβ toward VEGFR-2 signaling in cells. Moreover, Aβ was able to directly interact with the extracellular domain of VEGFR-2 and to compete with the binding of VEGF to its receptor in a cell-free assay. Co-immunoprecipitation experiments confirmed that Aβ can bind VEGFR-2 both in vitro and in vivo . Altogether, our data suggest that Aβ acts as an antagonist of VEGFR-2 and provide a mechanism explaining the anti-angiogenic activity of Aβ peptides.     

Essential involvement of the NMDA receptor in ethanol preconditioning-dependent neuroprotection from amyloid-βin vitro

In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-β (Aβ) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Aβ25–35 was undiminished by antagonism during the first 3 days of either adenosine A₁ or Gα_i/o protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Aβ neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR.     

Amyloid beta-derived neuroplasticity in bone marrow-derived mesenchymal stem cells is mediated by NPY and 5-HT2B receptors via ERK1/2 signalling pathways

Objective:  In Alzheimer's disease, toxic soluble and insoluble forms of amyloid beta (Aβ) cause synaptic dysfunction and neuronal loss. Given its potential role in producing a toxic host microenvironment for transplanted donor stem cells, we investigated the interaction between Aβ and proliferation, survival, and differentiation of bone marrow-derived mesenchymal stem cells (BM-MSC) in culture.
Materials and methods:  We used BM-MSC that had been isolated from mouse bone marrow and cultured, and we also assessed relevant reaction mechanisms using gene microarray, immunocytochemistry, and inhibitors of potential signalling molecules, such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2 and tyrosine protein kinase.
Results and conclusions:  Interestingly, we found that treatment with aggregated (1–40 or 1–42) and oligomeric (1–42) Aβ promoted neuronal-like differentiation of BM-MSC without toxic effects. This was not dependent on soluble factors released from BM-MSC progeny nor solely on formation of Aβ fibrils. The effect of Aβ is mediated by G-protein coupled receptors, neuropeptide Y1 (NPY1R) and serotonin (5-hydroxytryptamine) receptor 2B, via phosphatidylinositol-3-OH kinase-dependent activation of the MAPK/ERK1/2. Our results lend support to the idea that reciprocal donor stem cell–host interactions may promote a regenerative response that can be exploited by epigenetic modulation of NPY/serotonergic gene expression, for stem cell therapy, in Alzheimer's disease.     

Caffeine Stimulates Amyloid β-Peptide Release from β-Amyloid Precursor Protein-Transfected HEK293 Cells

Abstract: Extracellular amyloid β-peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor-based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 m M ) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated Aβ release. NH₄Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.     

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression. Special issue dedicated to Dr. Moussa Youdim.     

Apoptotic Cell Death Induced by β-Amyloid1–42 Peptide Is Cell Type Dependent

Abstract: β-Amyloid peptide (Aβ), a proteolytic fragment of the β-amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ_1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB₂a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ-induced cell death (necrosis or apoptosis) may depend on the cell type.     

Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells

Studies on amyloid beta (Aβ|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Aβ-mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Aβ on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Aβ_25–35 (10 μmol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 ± 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal Aβ_1–42 inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Aβ-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an Aβ-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease.