Francisco J. Muñoz

The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer’s disease

One of the theories involved in the etiology of Alzheimers disease (AD) is the oxidative stress hypothesis. The amyloid beta-peptide (A beta), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of different antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in A beta-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis.

Hemizygosity at the NCF1 Gene in Patients with Williams-Beuren Syndrome Decreases Their Risk of Hypertension

Williams-Beuren syndrome (WBS), caused by a heterozygous deletion at 7q11.23, represents a model for studying hypertension, the leading risk factor for mortality worldwide, in a genetically determined disorder. Haploinsufficiency at the elastin gene is known to lead to the vascular stenoses in WBS and is also thought to predispose to hypertension, present in approximately 50% of patients. Detailed clinical and molecular characterization of 96 patients with WBS was performed to explore clinical-molecular correlations. Deletion breakpoints were precisely defined and were found to result in variability at two genes, NCF1 and GTF2IRD2. Hypertension was significantly less prevalent in patients with WBS who had the deletion that included NCF1 (P=.02), a gene coding for the p47(phox) subunit of the NADPH oxidase. Decreased p47(phox) protein levels, decreased superoxide anion production, and lower protein nitrotyrosination were all observed in cell lines from patients hemizygous at NCF1. Our results indicate that the loss of a functional copy of NCF1 protects a proportion of patients with WBS against hypertension, likely through a lifelong reduced angiotensin II-mediated oxidative stress. Therefore, antioxidant therapy that reduces NADPH oxidase activity might have a potential benefit in identifiable patients with WBS in whom serious complications related to hypertension have been reported, as well as in forms of essential hypertension mediated by a similar pathogenic mechanism.

ESTROGEN PROTECTS NEURONAL CELLS FROM THE CYTOTOXICITY INDUCED BY ACETYLCHOLINESTERASE-AMYLOID COMPLEXES

The senile plaques present in Alzheimers disease (AD) are composed of a core of amyloid β‐peptide (Aβ) plus several proteins including acetylcholinesterase (AChE). Recently we found that AChE forms complexes with the Aβ peptide in vitro and that these are more cytotoxic than Aβ fibrils alone. Considering that estrogen has been reported to act as a protective agent against Aβ‐induced cytotoxicity, the effect of 17β‐estradiol was studied in rat pheochromocytoma (PC12) and mouse neuroblastoma (Neuro 2a) cells exposed to either Aβ alone or AChE‐Aβ complexes. Estrogen showed a powerful protective effect in response to the challenge of AChE‐Aβ complexes as well as with Aβ fibrils. This was also the case for other cytotoxic agents such as glutamate and H2O2. Our results suggest a common mechanism for cellular protection by estrogen against the toxicity of both Aβ fibrils and AChE‐Aβ complexes, likely avoiding the free radical apoptotic pathway.

Neurotoxicity of acetylcholinesterase amyloid β‐peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes

Alzheimers disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid β‐peptide (Aβ) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Aβ1–40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE‐Aβ complexes were found to be more toxic than those formed without the enzyme, for Aβ1–40 and Aβ1–42, but not for amyloid fibrils formed with AβVal18→Ala, a synthetic variant of the Aβ1–40 peptide. Of all the AChE‐Aβ complexes tested the one containing the Aβ1–40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Aβ1–40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE‐Aβ1–40 aggregates are more toxic than those of AChE‐Aβ1–42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation

Alzheimers disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimers disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimers disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimers disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimers disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimers disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimers disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid25-35 fragment, glutamate, and hydrogen peroxide.

This work addresses the differential effects of several oxidative insults on two neuronal cell lines, PC12 and Neuro 2a cells, extensively used as neuronal models in vitro. We measured cellular damage using the cytotoxic assays for MTT reduction and LDH release and found that acetylcholinesterase (AChE)–amyloid–β‐peptide (Aβ) complexes, Aβ25–35 fragment, glutamate and H2O2 were over 200‐fold more toxic to PC12 than to Neuro 2a cells. 17α and 17β estradiol were able to protect both cell types from damage caused by H2O2 or glutamate. By contrast, other insults not related to oxidative stress, such as those caused by the nonionic detergent Triton X‐100 and serum deprivation, induced a similar level of damage in both PC12 and Neuro 2a cells. Considering that the Aβ peptide, H2O2 and glutamate are cellular insults that cause an increase in reactive oxygen species (ROS), the intracellular levels of the antioxidant compound, glutathione were verified. Neuro 2a cells were found to have 4‐ to 5‐fold more glutathione than PC12 cells. Our results suggest that Neuro 2a cells are less susceptible to exposure to AChE–Aβ complexes, Aβ25–35 fragment, glutamate and H2O2 than PC12 cells, due to higher intracellular levels of antioxidant defense factors. J. Neurosci. Res. 56:620–631, 1999.u2003

The blood-brain barrier: Structure, function and therapeutic approaches to cross it

Abstract The blood-brain barrier (BBB) is constituted by a specialized vascular endothelium that interacts directly with astrocytes, neurons and pericytes. It protects the brain from the molecules of the systemic circulation but it has to be overcome for the proper treatment of brain cancer, psychiatric disorders or neurodegenerative diseases, which are dramatically increasing as the population ages. In the present work we have revised the current knowledge on the cellular structure of the BBB and the different procedures utilized currently and those proposed to cross it. Chemical modifications of the drugs, such as increasing their lipophilicity, turn them more prone to be internalized in the brain. Other mechanisms are the use of molecular tools to bind the drugs such as small immunoglobulins, liposomes or nanoparticles that will act as Trojan Horses favoring the drug delivery in brain. This fusion of the classical pharmacology with nanotechnology has opened a wide field to many different approaches with promising results to hypothesize that BBB will not be a major problem for the new generation of neuroactive drugs. The present review provides an overview of all state-of-the-art of the BBB structure and function, as well as of the classic strategies and these appeared in recent years to deliver drugs into the brain for the treatment of Central Nervous System (CNS) diseases.

Peripheral binding site is involved in the neurotrophic activity of acetylcholinesterase

Acetylcholinesterase (AChE) catalyses the hydrolysis of the neurotransmitter acetylcholine and it has been implicated in several non-cholinergic actions, including neurite outgrowth and amyloid formation. We have studied the trophic function of brain AChE on neuronal cell metabolism and proliferation as well as the enzyme domain involved in such effects. Low AChE concentrations (0.1-2.5 nM) stimulated neurite outgrowth and induced cell proliferation as measured by MTT reduction and [3H]thymidine incorporation. The action of AChE was not affected by edrophonium and tacrine both active site inhibitors, but it was abolished by propidium and gallamine, two peripheral anionic binding site (PAS) ligands. We conclude that the PAS domain of AChE is involved in the neurotrophic activity of the enzyme.

Oxidative stress triggers the amyloidogenic pathway in human vascular smooth muscle cells.

Cerebral amyloid angiopathy, associated to most cases of Alzheimers disease (AD), is characterized by the deposition of amyloid ss-peptide (Ass) in brain vessels, although the origin of the vascular amyloid deposits is still controversial: neuronal versus vascular. In the present work, we demonstrate that primary cultures of human cerebral vascular smooth muscle cells (HC-VSMCs) have all the secretases involved in amyloid ss-protein precursor (APP) cleavage and produce Ass(1-40) and Ass(1-42). Oxidative stress, a key factor in the etiology and pathophysiology of AD, up-regulates ss-site APP cleaving enzyme 1 (BACE1) expression, as well as Ass(1-40) and Ass(1-42) secretion in HC-VSMCs. This process is mediated by c-Jun N-terminal Kinase and p38 MAPK signaling and appears restricted to BACE1 regulation as no changes in the other secretases were observed. In conclusion, oxidative stress-mediated up-regulation of the amyloidogenic pathway in human cerebral vascular smooth muscle cells may contribute to the overall cerebrovascular amyloid angiopathy observed in AD patients.

Vitamin E But Not 17β-Estradiol Protects against Vascular Toxicity Induced by β-Amyloid Wild Type and the Dutch Amyloid Variant

Amyloid β-peptide (Aβ) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimers disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in Aβ yielding AβGlu22→Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated Aβ on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by Aβ1–40 Glu22→Gln and Aβ1–40 wild-type fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that AβGlu22→Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of Aβ fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. Aβ1–40 wild-type–AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of Aβ fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17β-estradiol on vascular damage induced by Aβwild-type and AβGlu22→Gln. Our data indicate that vitamin E attenuated significantly the Aβ-mediated cytotoxicity on vascular cells, although 17β-estradiol and vitamin C failed to inhibit the cytotoxicity induced by Aβ fibrils.