Orly Weinreb

Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases: Signaling pathways in EGCG neuroprotection

Accumulating evidence supports the hypothesis that brain iron misregulation and oxidative stress (OS), resulting in reactive oxygen species (ROS) generation from H2O2 and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinsons (PD), Alzheimers (AD) and Huntingtons diseases, and amyotrophic lateral sclerosis (ALS). Thus, novel therapeutic approaches aimed at neutralization of OS‐induced neurotoxicity, support the application of ROS scavengers, transition metals (e.g. iron and copper) chelators and non‐vitamin natural antioxidant polyphenols, in monotherapy, or as part of antioxidant cocktail formulation for these diseases. Both experimental and epidemiological evidence demonstrate that flavonoid polyphenols, particularly from green tea and blueberries, improve age‐related cognitive decline and are neuroprotective in models of PD, AD and cerebral ischemia/reperfusion injuries. However, recent studies indicate that the radical scavenger property of green tea polyphenols is unlikely to be the sole explanation for their neuroprotective capacity and in fact, a wide spectrum of cellular signaling events may well account for their biological actions. In this article, the currently established mechanisms involved in the beneficial health action and emerging studies concerning the putative novel molecular neuroprotective activity of green tea and its major polyphenol (‐)‐epigallocatechin‐3‐gallate (EGCG), will be reviewed and discussed.

Regulation of Bcl-2 family proteins, neurotrophic factors, and APP processing in the neurorescue activity of propargylamine

The anti‐Parkinson drug, rasagiline (N‐propargyl‐(1R)‐aminoindan) promotes neuronal survival, via neuroprotective activity related to its propargyl moiety (propargylamine). We have investigated the neurorescue effects of propargylamine, in a progressive neuronal death model, induced by long‐term serum deprivation in human SH‐SY5Y neuroblastoma cells. Propargylamine (0.1–10 µM) dose‐dependently reduced the levels of the early apoptosis‐associated phosphorylated protein, H2A‐X (ser 139), as well as decreased the cleavage of caspase‐3 and its substrate poly‐ADP ribose polymerase (PARP). In addition, the compound markedly reversed the apoptotic effects induced by long‐term serum withdrawal, including down‐regulation of the antiapoptotic protein, Bcl‐2, as well as up‐regulation of the proapoptotic proteins, Bax, Bad, and Bim. Real‐time RT‐PCR demonstrated that propargylamine elevated gene expression levels of Bcl‐2, and the neurotrophic factors glial cell line‐derived neurotrophic factor (GDNF) and brain‐derived neurotrophic factor (BDNF) and reduced Bax gene expression. Serum deprivation increased mRNA and protein levels of holo‐amyloid precursor protein (APP), which was markedly decreased by propargylamine. This was accompanied by inducing the release of the nonamyloidogenic α‐secretase form of soluble APP (sAPPα) into the medium. Similar effects on cell survival and APP regulation/processing were demonstrated for rasagiline. These results indicate that both rasagiline and propargylamine possess neurorescue activity, associated with regulation of Bcl‐2 family proteins, neurotrophic factors, and APP metabolism.

Rasagiline: Neurodegeneration, neuroprotection, and mitochondrial permeability transition

Mitochondria are involved directly in cell survival and death. The assumption has been made that drugs that protect mitochondrial viability and prevent apoptotic cascade‐induced mitochondrial permeability transition pore (MPTp) opening will be cytoprotective. Rasagiline (N‐propargyl‐1R‐aminoindan) is a novel, highly potent irreversible monoamine oxidase (MAO) B inhibitor anti‐Parkinson drug. Unlike selegiline, it is not derived from amphetamine, and is not metabolized to neurotoxic L‐methamphetamine derivative. In addition, it does not have sympathomimetic activity. Rasagiline is effective as monotherapy or adjunct to levodopa for patients with early and late Parkinsons disease (PD) and adverse events do not occur with greater frequency in subjects receiving rasagiline than in those on placebo. Phase III controlled studies indicate that it might have a disease‐modifying effect in PD that may be related to its neuroprotective activity. Its S isomer, TVP1022, is more than 1,000 times less potent as an MAO inhibitor. Both drugs, however, have neuroprotective activity in neuronal cell cultures in response to various neurotoxins, and in vivo in response to global ischemia, neurotrauma, head injury, anoxia, etc., indicating that MAO inhibition is not a prerequisite for neuroprotection. Their neuroprotective effect has been demonstrated to be associated directly with the propargylamine moiety, which protects mitochondrial viability and MTPp by activating Bcl‐2 and protein kinase C (PKC) and by downregulating the proapoptotic FAS and Bax protein families. Rasagiline and its derivatives also process amyloid precursor protein (APP) to the neuroprotective, neurotrophic, soluble APP α (sAPPα) by PKC‐ and MAP kinase‐dependent activation of α‐secretase. The identification of the propargylamine moiety as the neuroprotective component of rasagiline has led us to development of novel bifunctional anti‐Alzheimer drugs (ladostigil) possessing cholinesterase and brain‐selective MAO inhibitory activity and a similar neuroprotective mechanism of action.

Cell Signaling Pathways and Iron Chelation in the Neurorestorative Activity of Green Tea Polyphenols: Special Reference to Epigallocatechin Gallate (EGCG)

Although much progress has been made in understanding the pathogenesis of Alzheimers disease (AD), the current therapeutic approaches are merely symptomatic, intended for the treatment of cognitive symptoms, such as disturbances in memory and perception. Novel promising strategies suggest the use of anti-inflammatory drugs, antioxidants including natural occurring plant flavonoids, iron-complexing molecules, neurotrophic factor delivery, inhibitors of the amyloid-beta protein precursor processing secretases, gamma and beta, that generate amyloid-beta peptides and the interference with lipid and cholesterol metabolism. Human epidemiological and new animal data suggest that tea drinking may decrease the incidence of dementia, AD and Parkinsons disease. In particular, its main catechin polyphenol constituent (-)-epigallocatechin-3-gallate (EGCG) has been shown to exert neuroprotective/neurorescue activities in a wide array of cellular and animal models of neurological disorders. This review provides a detailed overview on the multimodal activities of green tea polyphenols with emphasis on their iron chelating, neurorescue/neuroregenerative and mitochondrial stabilization action.

cDNA gene expression profile homology of antioxidants and their antiapoptotic and proapoptotic activities in human neuroblastoma cells

Antioxidants have concentration‐dependent neuroprotective and proapoptotic activities in models of Parkinsons disease. The aim of our study was to determine gene‐protein pathways of the antioxidants, dopamine (DA), R‐apomorphine (R‐APO), melatonin, and green tea polyphenol (−)‐epigallocatechin‐3‐gallate (EGCG), in neuroblastoma cells, using a customized cDNA microarray and quantitative reverse transcriptase‐polymerase chain reaction gene expression techniques. We demonstrate a concentration‐dependent correlation between these compounds and modulation of cell survival/cell death‐related gene pathways. High toxic concentration of DA (500 µM), R‐APO (50 µM), melatonin (50 µM), and EGCG (50 µM) exhibited a similar profile of proapoptotic gene expression, increasing the level of bax, caspase‐6, fas ligand, and the cell‐cycle inhibitor gadd45 genes, while decreasing antiapoptotic bcl‐2 and bcl‐xL. Conversely, the low neuroprotective concentrations (1−10 µM) of these compounds induced an antiapoptotic response. Melatonin displayed an extremely low index of mortality, which may be partially explained by the observation that a high concentration did not significantly affect the expression of mitochondrial Bcl‐2 family members, bcl‐2 and bax. Protein analysis of Bcl‐2, Bax, and activated caspase‐3 correlated with the gene expression pattern. Our results provide for the first time new insights into the molecular events involved in the dose‐dependent neuroprotective and neurotoxic activities of catechols and indole amine compounds.

Neurorescue Activity, APP Regulation and Amyloid-β Peptide Reduction by Novel Multi-Functional Brain Permeable Iron- Chelating- Antioxidants,M-30 and Green Tea Polyphenol, EGCG

Accumulation of iron at sites where neurons degenerate in Parkinsons disease (PD) and Alzheimers disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine-1-ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5-untranslated region (5UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP Swedish mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntingtons disease and amyotrophic lateral sclerosis.

Induction of Neurotrophic Factors GDNF and BDNF Associated with the Mechanism of Neurorescue Action of Rasagiline and Ladostigil

Parkinsons disease (PD) and Alzheimers disease (AD) are the most common neurodegenerative disorders, although there is no drug or therapeutic treatment to demonstrate disease‐modifying effects. Previous work has proposed that neurodegeneration is linked to a lack of trophic support in those neurons and brain areas associated with PD and AD. Indeed, previous studies have found that neurotrophic factors (NTFs) support neuronal survival in various cellular and animal models of PD and AD. Thus, attention has begun to turn to the possibility of NTF neuroprotective–neurorescue therapies for these diseases, indicating that NTFs may be of significant clinical importance as exogenously supplied or endogenously induced elements that obliterate neuronal deficits and degeneration. We have recently reported that the anti‐PD drug rasagiline, the anti‐AD drug ladostigil, and their propargyl moiety, propargylamine, enhanced the expression levels of brain‐derived neurotrophic factor and glial cell line–derived neurotrophic factor, endogenous NTFs associated with activation of phosphatidylinositol 3‐kinase, protein kinase, and mitogen‐activated protein kinase cell signaling/survival pathways. These studies indicate that the induction of NTFs by rasagiline and ladostigil might suppress apoptosis and induce neurorescue in neurodegenerative disorders and may support the drugs possible disease‐modifying mechanism of action.

Ladostigil: A Novel Multimodal Neuroprotective Drug with Cholinesterase and Brain-Selective Monoamine Oxidase Inhibitory Activities for Alzheimers Disease Treatment

Ladostigil [(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate] is a dual acetylcholine-butyrylcholineesterase and brain selective monoamine oxidase (MAO)-A and -B inhibitor in vivo (with little or no MAO inhibitory effect in the liver and small intestine), intended for the treatment of dementia co-morbid with extrapyramidal disorders and depression (presently in a Phase IIb clinical study). This suggests that the drug should not cause a significant potentiation of the cardiovascular response to tyramine, thereby making it a potentially safer antidepressant than other irreversible MAO-A inhibitors. Ladostigil was shown to antagonize scopolamine-induced impairment in spatial memory, indicating that it can cause significant increases in rat brain cholinergic activity. Furthermore, ladostigil prevented gliosis and oxidative-nitrative stress and reduced the deficits in episodic and spatial memory induced by intracerebroventricular injection of streptozotocin in rats. Ladostigil was demonstrated to possess potent anti-apoptotic and neuroprotective activities in vitro and in various neurodegenerative rat models, (e.g. hippocampal damage induced by global ischemia in gerbils and cerebral oedema induced in mice by closed head injury). These neuroprotective activities involve regulation of amyloid precursor protein processing; activation of protein kinase C and mitogen-activated protein kinase signaling pathways; inhibition of neuronal death markers; prevention of the fall in mitochondrial membrane potential and upregulation of neurotrophic factors and antioxidative activity. Recent findings demonstrated that the major metabolite of ladostigil, hydroxy-1-(R)-aminoindan has also a neuroprotective activity and thus, may contribute to the overt activity of its parent compound. This review will discuss the scientific evidence for the therapeutic potential use of ladostigil in Alzheimers and Lewy Body diseases and the molecular signaling pathways that are considered to be involved in the biological activities of the drug.

Neuroprotective Multifunctional Iron Chelators: From Redox-Sensitive Process to Novel Therapeutic Opportunities

Accumulating evidence suggests that many cytotoxic signals occurring in the neurodegenerative brain can initiate neuronal death processes, including oxidative stress, inflammation, and accumulation of iron at the sites of the neuronal deterioration. Neuroprotection by iron chelators has been widely recognized with respect to their ability to prevent hydroxyl radical formation in the Fenton reaction by sequestering redox-active iron. An additional neuroprotective mechanism of iron chelators is associated with their ability to upregulate or stabilize the transcriptional activator, hypoxia-inducible factor-1alpha (HIF-1alpha). HIF-1alpha stability within the cells is under the control of a class of iron-dependent and oxygen-sensor enzymes, HIF prolyl-4-hydroxylases (PHDs) that target HIF-1alpha for degradation. Thus, an emerging novel target for neuroprotection is associated with the HIF system to promote stabilization of HIF-1alpha and increase transcription of HIF-1-related survival genes, which have been reported to be regulated in patients brains afflicted with diverse neurodegenerative diseases. In accordance, a new potential therapeutic strategy for neurodegenerative diseases is explored, by which iron chelators would inhibit PHDs, target the HIF-1-signaling pathway and ultimately activate HIF-1-dependent neuroprotective genes. This review discusses two interrelated approaches concerning therapy targets in neurodegeneration, sharing in common the implementation of iron chelation activity: antioxidation and HIF-1-pathway activation.

Gene and Protein Expression Profiles of Anti‐ and Pro‐apoptotic Actions of Dopamine, R‐Apomorphine, Green Tea Polyphenol (−)‐Epigallocatechine‐3‐gallate, and Melatonin

Abstract: Significant evidence has been provided to support the hypothesis that oxidant stress may be responsible for degeneration of dopaminergic neurons in the substantia nigra pars compacta in Parkinsons disease. Dopamine (DA), R‐apomorphine (R‐APO), green tea polyphenol (−)‐epigallocatechine‐3‐gallate (EGCG), and melatonin are neuroprotective and radical scavenger compounds. The aim of this study was to establish the mechanism of the concentration‐dependent neuroprotective and pro‐apoptotic action of these drugs via gene expression and protein determination. cDNA microarrays provide new prospects to study and identify various mechanisms of drug action. We employed this technique for the study reported in this paper. Total RNA was extracted from SH‐SY5Y cells exposed to low neuroprotective and high toxic concentrations of the drugs, followed by synthesis of cDNA, and hybridization to a microarray membrane related to apoptosis, survival, and cell cycle pathways. We demonstrated a concentration and time‐dependent correlation between R‐APO, DA, EGCG, and melatonin in modulation of cell survival/cell death‐related gene pathways. The results were confirmed by quantitative real‐time PCR and protein profiles. Unlike the effects of low concentrations (1‐10 μM), where an antiapoptotic response was manifest, a proapoptotic pattern of gene expression was observed at high toxic concentrations (50‐500 μM) of the antioxidants (e.g., increase in caspases, fas, and gadd45). Our results have provided novel insights into the gene mechanisms involved in both the neuroprotective and proapoptotic activities of neuroprotective drugs. We have shown that DA, R‐APO, EGCG, and melatonin exhibit similar gene expression and protein profiles.