APOE3 Christchurch modulates tau phosphorylation and ß-catenin/Wnt/Cadherin signaling in induced pluripotent stem cell-derived cerebral organoids from Alzheimer's cases.

Alzheimer's disease (AD) is the most common cause of dementia among older adults. APOE3 Christchurch (R136S, APOE3Ch ) variant homozygosity was reported in an individual with extreme resistance to autosomal dominant AD due to the PSEN1 E280A mutation. This subject had a delayed clinical age at onset and resistance to tauopathy and neurodegeneration despite extremely high amyloid plaque burden. We established induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and from a non-protected kindred control (with PSEN1 E280A and APOE3/3 ). We used CRISPR/Cas9 gene editing to successfully remove the APOE3Ch to wild type in iPS cells from the protected case and to introduce the APOE3Ch as homozygote in iPS cells from the non-protected case to examine causality. We found significant reduction of tau phosphorylation (pTau 202/205 and pTau396) in cerebral organoids with the APOE3Ch variant, consistent with the strikingly reduced tau pathology found in the resistant case. We identified Cadherin and Wnt pathways as signaling mechanisms regulated by the APOE3Ch variant through single cell RNA sequencing in cerebral organoids. We also identified elevated ß-catenin protein, a regulator of tau phosphorylation, as a candidate mediator of APOE3Ch resistance to tauopathy. Our findings show that APOE3Ch is necessary and sufficient to confer resistance to tauopathy in an experimental ex-vivo model establishing a foundation for the development of novel, protected case-inspired therapeutics for tauopathies, including Alzheimer's.

N-acetyl-cysteine blunts 6-hydroxydopamine- and L-buthionine-sulfoximine-induced apoptosis in human mesenchymal stromal cells.

Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10 mM) plus 6-OHDA (200 µM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5 mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.

PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine-induced apoptosis: Potential therapeutic strategy in Parkinson's disease.

BACKGROUND AIMS: Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. METHODS: We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. RESULTS: After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). DISCUSSION: 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.

Fast transdifferentiation of human Wharton's jelly mesenchymal stem cells into neurospheres and nerve-like cells.

BACKGROUND: The human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs) represent a tool for cell-based therapies and regenerative medicine. hWJ-MSCs form neurospheres (NSs) within 3-7 days. No data is available to establish the neuro-phenotypic markers and time of formation of nerve-like (NLCs) and glial cells from NSs derived from hWJ-MSCs. NEW METHOD: hWJ-MSCs were incubated with Fast-N-Spheres medium for 24 and 72h. The new formed NSs were in turn incubated with forskolin in neurogenic NeuroForsk medium for 1-7days. RESULTS: hWJ-MSCs cultured with Fast-N-Spheres medium trans-differentiated into NSs in just 24h compared to 72h for hWJ-MSCs cultured with classic growth factor medium. The NSs generated from the Fast-N-Spheres medium expressed reduced levels SOX2, OCT4 and NANOG, as markers of pluripotency compared to undifferentiated hWJ-MSCs. The formed NSs exposed to NeuroForsk medium differentiated into NLCs in 4days as evidenced by high levels of protein expression of the neuronal markers, and no expression of the glial marker GFAP. COMPARISON WITH EXISTING METHOD(S): Currently, the formation and harvest of NSs is expensive and time consuming. Published protocols require 3-7days to form NSs from whole human umbilical cord MSCs. We report for the first time, to our knowledge, the differentiation of NSs-derived from hWJ-MSCs into NLCs. CONCLUSIONS: The fastest method to obtain NSs and NLCs from hWJ-MSCs takes only five days using the two-step incubation media Fast-N-Spheres and NeuroForsk.

The effects of polyphenols on survival and locomotor activity in Drosophila melanogaster exposed to iron and paraquat.

Parkinson's disease (PD) is a common progressive neurodegenerative disorder, for which at present no causal treatment is available. On the understanding that the causes of PD are mainly oxidative stress and mitochondrial dysfunction, antioxidants and other drugs are expected to be used. In the present study, we demonstrated for the first time that pure polyphenols such as gallic acid, ferulic acid, caffeic acid, coumaric acid, propyl gallate, epicatechin, epigallocatechin, and epigallocatechin gallate protect, rescue and, most importantly, restore the impaired movement activity (i.e., climbing capability) induced by paraquat in Drosophila melanogaster, a valid model of PD. We also showed for the first time that high concentrations of iron (e.g. 15 mM FeSO(4)) are able to diminish fly survival and movement to a similar extent as (20 mM) paraquat treatment. Moreover, paraquat and iron synergistically affect both survival and locomotor function. Remarkably, propyl gallate and epigallocatechin gallate protected and maintained movement abilities in flies co-treated with paraquat and iron. Our findings indicate that pure polyphenols might be potent neuroprotective agents for the treatment of PD against stressful stimuli.

The cannabinoid CP55,940 prolongs survival and improves locomotor activity in Drosophila melanogaster against paraquat: implications in Parkinson's disease.

Cannabinoids have been shown to function as protective agents via receptor-independent and/or receptor-dependent mechanisms against stressful conditions. However, the neuroprotective mechanism of cannabinoids is far from conclusive. Therefore, the genuine antioxidant impact of cannabinoids in vivo is still uncertain. In this study, we demonstrate for the first time that CP55,940, a nonselective CB(1)/CB(2) cannabinoid receptor agonist, significantly protects and rescues Drosophila melanogaster against paraquat (PQ) toxicity via a receptor-independent mechanism. Interestingly, CP55,940 restores the negative geotaxis activity (i.e., climbing capability) of the fly exposed to PQ. Moreover, Drosophila fed with (1-200 microM) SP600125, a specific inhibitor of the stress responsive Jun-N-terminal kinase (JNK) signaling, and 20 mM PQ increased survival percentage and movement function (i.e., climbing capability) when compared to flies only treated with PQ. Taken together our results suggest that exogenous antioxidant cannabinoids can protect against and rescue from locomotor dysfunction in wild type (Canton-S) Drosophila exposed to stress stimuli. Therefore, cannabinoids may offer promising avenues for the design of molecules to prevent, delay, or ameliorate the treatment of population at high risk of suffering Parkinson disease.

A new method of standartization of health-promoting pomegranate fruit (Punica granatum) extract.

This study analyzes the major phenolic constituents of pomegranate fruit juice and pericarp feedstock, and dry extracts thereof, using high-performance liquid chromatography. Pomegranate pericarp was extracted with water (WE) and alcohol (AE) as solvents, and liquid extracts were subsequently freeze-dried. The results indicate that ellagitannins punicalagin A and punicalagin B are the major constituents in the primary pomegranate feedstock and in both types of extracts. Ellagic acid, a common botanical constituent that is currently used to standardize pomegranate extracts, as well as ellagitannin punicalin, were found to be only minor constituents. Total punicalagins (the sum of punicalagins A+B and punicalin) and ellagic acid content in the pomegranate fruit pericarp feedstock WE were 7,6+/-0,3% and 0,2+/-0,1% by dry weight, respectively, and in the AE feedstock 7,0+/-0,2 and 0,4+/-0,1%, respectively. Total phenolic content (the sum of punicalagins and ellagic acid) in the pomegranate WE and AE were 45,8+/-1,2% and 42,3+/-1,1%, respectively. The concentrations of ellagic acid in the pomegranate WE and AE were 0,8+/-0,2% and 3,9+/-0,2%, respectively. Total phenolics in fresh single-strength pomegranate whole fruit juice contained 2,216+/-70 mg/L (95% punicalagins), whereas commercial pomegranate juice that was purchased from local stores was 317+/-13 mg/L (70% punicalagins). Our results strongly suggest that the commercially produced pomegranate extracts should be standardized to the content of total punicalagins as well as ellagic acid. The current standard uses only ellagic acid, which is unreliable, potentially misleading and vulnerable to commercial adulteration.

Efficiency of pharmacologically-active antioxidant phytomedicine Radical Fruits in treatment hypercholesteremia at men.

The purpose of this randomized, double-blind, placebo controlled intervention clinical trial was to investigate the effect of orally administered dietary supplement Radical Fruits (www.gardenoflife.com) consisting of highly concentrated edible fruits and berries on the concentration of plasma cholesterol, urinary 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydrothromboxane B2 (11-dehydro-TXB2) in none-obese, non-smoking, non-diabetic hypercholesteremic male volunteers. Forty four (n=44) none-obese, non-smoking, non-diabetic male volunteers with an average age of 40+/-12 years, average body weight of 77,4+/-5,0 kg, average body mass index (BMI) of 22,2+/-2,7 kg/m2 and average total plasma cholesterol level 280+/-22 mg/dL were recruited to take part in a 4 week double-blind, randomized, placebo-controlled clinical trial. After evaluating 12 different antioxidant supplements for their phytochemical compositions and bioavailability, a dietary supplement Radical Fruits was chosen for this clinical trial. Radical Fruits contains standardized extracts and concentrates of prune, pomegranate, apple, grape, raspberry, blueberry, white cherry and strawberry. Subjects were randomly assigned to the treatment group and the placebo group using Simple Randomization Procedure. Subjects in the treatment group (n=22) were directed to take 900 mg of Radical Fruits supplement three times a day before meals. Subjects in the control group (n=22) were directed to take placebo according to the same schedule. Food record analysis, body composition, blood and urine samples were assessed on admission and then once a week. The duration of the clinical trial was 4 weeks. Administration of Radical Fruits(TM) for 4 weeks resulted in statistically significant reduction of total plasma cholesterol from 280+/-23 to 250+/-11 mg/dL, (p<0,001). Total plasma cholesterol changes in the placebo group were not statistically significant. The average plasma LDL was reduced from 195+/-23 to 169+/-21 mg/dL (p<0,001) in the Radical Fruits(TM) group, while in the placebo group there were no statistically significant changes. Plasma HDL increased by 3,2=/-0,6% in the Radical Fruits treated group (p<0,001). No significant changes in the HDL levels were observed in the placebo group. Urinary 8-epi-PGF2alpha level decreased from 450+/-170 to 330+/-159 pg/mg creatinine (p<0,001); urinary 11-dehydro-TXB2 level decreased from 1,200+/-420 to 790+/-320 pg/mg creatinine (p<0,001) with no changes in the placebo group. Administration of pharmacologically active antioxidant supplement Radical Fruit(TM) in hypercholesteremic men significantly increased plasma HDL and reduced total cholesterol and LDL, and urinary oxidative and inflammatory isoprostanes and thromboxane.

Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study.

In a 4-week randomized placebo-controlled clinical trial we investigated the effect of 300 mg Blueberin, a phytomedicine containing 250 mg Blueberry leaves (Vaccinium arctostaphylos L, Ericaceae) extract providing minimum 50 mg 3,4-caffeoylquinic (chlorogenic) acid, and 50 mg myricetin, on fasting plasma glucose, alanine aminotransferases (ALT), aspartate aminotransferases (AST), glutamyltransferase (GGT) enzymes levels, and serum inflammatory C-Reactive proteins (CRP) in forty-two volunteer subjects (46+/-15 year of age, BMI 25+/-3 kgs/(m2)) diagnosed with Type 2 diabetes. During the 4-week trial, the Blueberin supplement was administered three times per day, 15-30 minutes prior to a meal along with 100 ml of water. Results of this trial revealed that the supplementation of Blueberin reduced fasting plasma glucose from 143+/-5,2mg/L to 104+/-5,7 mg/L (p<0,001), whereas there was no statistically significant changes in the Placebo group from 138+/-4,8 mg/L to 126+/-5,1mg/L (p>0,05). The reduction of fasting glucose was correlated with the reduction of serum CRP and in the Blueberin group from 5,18+/-1,4 mg/l to 2,14+/-1,8 mg/L (p<0,05), whereas in the Placebo group CRP levels were not significantly reduced from 5,11+/-1,7 mg/l to 4,94+/-1,1mg/L (p>0,05). Furthermore, the Blueberin also significantly reduced the levels of plasma enzymes ALT, AST and GGT, indicating that, in addition to anti-diabetes effects, the Blueberin also possess pharmacologically relevant anti-inflammatory properties.

[Neuronal DNA damage correlates with a positive detection of c-Jun, nuclear factor kB, p53 and Par-4 transcription factors in Alzheimer's disease]. / El daño nuclear neuronal se correlaciona con la detección in situ de los factores de transcripción c-Jun, factor nuclear kB, p53 y Par-4 en la enfermedad de Alzheimer.

INTRODUCTION AND OBJECTIVES: Alzheimer s disease is a neurodegenerative disorder characterized neuropathologically by beta amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. Recently, we have elucidated a molecular cascade of cell death induced by A beta 25 35 involving the activation of nuclear factor kappa B (NF kB), p53, and c Jun transcription factors in vitro. At present, no comparative reports have been published to establish a similar cell death signalization pathway in in vitro and in in vivo. The aim of this investigation was to determine if AD brains might activate NF kB, p53, c Jun, Par 4 transcription factors and to establish whether there exist a relationship between neuronal DNA damage and transcription factors activation. PATIENTS AND METHODS: We investigated Ab plaques, neurofibrillary tangles and NF kB, p53, and c Jun transcription factor activation in five cerebral regions from 3 normal subjects and from six demented patient with sporadic AD and one patient with AD familiar according to CERAD criteria. Using TUNEL we determine neuronal damage. RESULTS: We demonstrated neuronal damage in 17 out of 50 regions evaluated as TUNEL positive, and their distribution was heterogeneous in all brain regions evaluated; and the activation of NF kB, p53, c Jun and Par 4 transcription factors from case # 24 and #22, corresponding to TUNEL positive. CONCLUSIONS: We found a correlation between severity of DNA damage and nuclear activation of the transcription factors. These findings suggest that the AD brain may induce cell death by a molecular signalization similar to a non neuronal model by Ab exposure. This in situ study might validate previous Ab induced cell death observations in vitro.

El daño nuclear neuronal se correlaciona con la detección in situ de los factores de transcripción c-Jun, factor nuclear B, p53 y Par-4 en la enfermedad de Alzheimer / Neuronal DNA damage correlates with a positive detection of c-Jun, nuclear factor kB P53 and Par-4 transcription factors in Alzheimer´s disease

Introducción y objetivos. La enfermedad de Alzheimer (EA) se caracteriza neuropatológicamente por placas de Beta-amiloide, ovillos neurofibrilares (ONF), gliosis y pérdida neuronal. Recientemente, hemos elucidado una cascada de muerte celular inducida por el Alfa-Beta A25-35 que involucra la activación de los factores de transcripción nuclear B (FN-B), p53 y c-Jun in vitro. Actualmente, no se han comunicado estudios comparativos entre los eventos moleculares de muerte celular in vitro e in vivo. El objetivo fue determinar si en los cerebros con EA se activan el FN-kB, p53, c-Jun y Par-4,y establecer si esta activación se relaciona con la fragmentación del ADN neuronal. Pacientes y métodos. Se investigó la presencia de placas, ONF y activación de los factores de transcripción en cinco regiones de tejido post mortem en tres cerebros de sujetos normales, en seis pacientes diagnosticados de Alzheimer esporádico y en uno dignosticado de Alzheimer familiar con los criterios de CERAD. El daño neuronal se determinó con TUNEL. Resultados. Evidenciamos daño neuronal en 17 de 50 regiones analizadas positivas para TUNEL, con una distribución heterogénea en todas las regiones evaluadas. También se puso de manifiesto la activación del FN-kB, p53, Par-4 y c-Jun en los casos n.º 22 y 24, correspondientes a las regiones positivaspara TUNEL. Conclusión. Demostramos una correlación entre la gravedad del daño neuronal y la activación nuclear de los factores de transcripción. Estos hallazgos sugieren que en los cerebros afectados de EA se podría inducir muerte neuronal por una señalización molecular similar a modelos no neuronales por exposición al A25-35. Este estudio in situ valida observaciones previas de muerte celular inducida por el A25-35 in vitro (AU)

[Apoptosis in neurodegenerative diseases: facts and controversies]. / La apoptosis en las enfermedades neurodegenerativas: evidencias y controversias.

OBJECTIVES: In this article, the authors analyzed critically the morphological and biochemical evidences of cell death by apoptosis from postmortem studies in Alzheimer s, Parkinson s, Huntington s and Wilson s diseases. DEVELOPMENT: During the last few years, apoptosis has been postulated as a type of neuronal death responsible for the neurodegenerative process in those heterogeneous, chronic and progressive neurological disorders, which are characterized by a selective and a symmetric loss of neurons in motor, sensory or cognitive systems. With regard to neuronal death mechanism and the contribution of the mutated or metabolic altered proteins such as betaA, P-tau, alpha-synuclein, Parkin, Huntingtin, ATP78B, proteins in the pathogenesis of those disorders are still unknown. CONCLUSIONS: We consider that the morphological (e.g. DNA fragmentation without showing classical apoptotic morphology) and biochemical evidences are still insufficient and contradictory to formally indict apoptosis as the mechanism of neuronal cell death in those neurological disorders. The establishment of the molecular mechanisms leading neurons to cell death (by apoptosis?) could provide significant information for the design of therapeutic strategies to retard or prevent the development of such neurodegenerative diseases in affected individuals.

Screening of antimicrobial activities in red, green and brown macroalgae from Gran Canaria (Canary Islands, Spain).

Extracts from 44 species of seaweed from Gran Canaria (Canary Islands, Spain) were screened for the production of antibacterial and antifungal compounds against a panel of gram-negative and gram-positive bacteria, mycobacteria, yeasts and fungi. A total of 28 species displayed antibacterial activity, of which six also showed antifungal activity. Asparagopsis taxiformis and Cymopolia barbata were the species with the strongest activities against the broadest spectrum of target microorganisms. All the species with antibacterial activity were active against gram-positive bacteria, whereas only two species, A. taxiformis and Osmundea hybrida, were active against mycobacteria. The production of secondary metabolites with antimicrobial activities by the macroalgae was also studied under different conditions, although no common trend for bioactivity was observed.

Molecular mechanism of monoamine toxicity in Parkinson's disease: hypothetical cell death model.

Although there have been experimental approaches to understanding the etiology of Parkinson's disease, the cause of cell degeneration in this neurological disorder remains a mystery. Herein, a hypothetical model is proposed to explain the mechanism leading neurons to die. The model is based on recent experimental evidence and it attempts to dissect the actions of dopamine and metal ions as potential triggers for the activation of an ordered cascade of events of the cell death machinery.

DNA Damage does not Correlate with Amyloid-beta-Plaques and Neurofibrillary Tangles in Familial Alzheimer's Disease Presenilin-1 [E280A] Mutation.

Recent studies have shown that the missense mutation in presenilin-1 [E280A] increases deposition of amyloid-beta (Abeta) and neurofibrillary tangle (NFT) formation. Indeed, we only observed 10 out of 48 FAD brain sections displaying TUNEL (+) labeling, and none with the classical apoptotic morphology. These results may indicate that DNA fragmentation is not a generalized phenomenon in early-onset FAD PS1 [E280A] patients or that neuronal cells are dying by a different mechanism of cell death. Taking together these findings suggest that Abeta and NFTs are not per se a causative factor to damage neuronal cells but their damage could be more related with individual neuronal vulnerability and brain aging

17 beta-estradiol protects lymphocytes against dopamine and iron-induced apoptosis by a genomic-independent mechanism. Implication in Parkinson's disease.

Dopamine (DA) in combination with iron (Fe(2+)) has been demonstrated to induce apoptosis in neuronal-like PC12 cells by an oxidative stress mechanism. To get a better insight of cell death and protective mechanisms in DA/Fe(2+)-induced toxicity, we investigated the effects of DA/Fe(2+) and the antioxidant action of 17 beta-estradiol (E2) in peripheral blood lymphocytes (PBL). We found that DA/Fe(2+)-induces apoptosis in PBL via a hydrogen peroxide (H(2)O(2))-mediated oxidative mechanism, which in turn triggers a cascade of molecular events requiring RNA and de novo protein synthesis. We have also demonstrated that E2 prevents significantly DA/Fe(2+)-induced apoptosis in PBL by directly inhibiting the intracellular accumulation of peroxides generated by DA/Fe(2+)-reaction. This protective activity is independent of the presence or activation of the estrogen receptors (ERs). These data further support and validate our previous hypothesis that DA/Fe(2+)/H(2)O(2) could be a general mediator of oxidative stress through a common cell death mechanism in both neuronal and nonneuronal cells. These findings may be particularly relevant to the potential approaches to rescue and prolong the survival of neurons by estrogens in patients with Parkinson's disease (PD).

Familial Alzheimer's disease: oxidative stress, beta-amyloid, presenilins, and cell death.

1. The basic etiology of Alzheimer's disease remains unknown, although four genes have so far been involved: beta-amyloid precursor protein, presenilin-1, presenilin-2 and apolipoprotein E genes. 2. The largest familial Alzheimer's disease (FAD) kindred so far reported belong to a point mutation in codon 280 that results in a glutamic acid-to-alanine substitution in presenilin-1 characterized in Antioquia, Colombia. 3. A hypothetical unified molecular mechanism model of cell death in FAD mediated by presenilin-1, beta-amyloid, and oxidative stress is proposed as an attempt to explain the mechanisms of neuronal loss in this neurodegenerative disorder.

Dopamine and iron induce apoptosis in PC12 cells.

Recent studies have shown that Fe2+ increases the oxidation of monoamines such as serotonin, dopamine and related toxins and that the formed oxidation products can undergo co-valent binding to free sulphydryl groups of proteins such as actin and "serotonin binding proteins" which are present in soluble brain extracts. Here we have tested the ability of ferrous iron to induce [3H]dopamine association to cytoplasmic proteins and we have established that a similar oxidation mechanism evidenced in vitro studies could be applied in cell culture. When PC12 cells were incubated with ferrous iron (ferrocene), the binding of [3H]dopamine to proteins was found to be two fold increased with respect to control. The iron is likely to accelerate the oxidation of dopamine to produce quinones which covalently bind to proteins and induce high-molecular protein aggregates. We evidenced that dopamine/iron combination induced cell death in undifferentiated PC12 cells via an active cellular process evaluated in terms of morphological and biochemical changes indicative of apoptosis. We also demonstrated induction of lipid peroxidation when dopamine and ferrocene were present in high concentrations. Moreover, ascorbic acid diminished apoptosis but not the lipid peroxidation process. It might indicate that ferrocene and dopamine could produce oxidative stress of a different nature. These results show that the actions of dopamine and iron are essential in the induction of apoptosis and lipid peroxidation. However, there is no necessary casual link between lipid peroxidation and apoptosis. Our data also suggest that iron is capable of increasing the cytotoxicity of dopamine merely by increasing its rate of oxidation and without intervention of the monoamine oxidase B enzyme and, hence, both phenomenons may occur independently from each other in rat pheochromocytoma PC12. These observations may have relevance to the understanding of the mechanism by which dopaminergic neurones are destroyed in some neurodegenerative disorders.