Orit Bar-Am

Neuroprotection via pro-survival protein kinase C isoforms associated with Bcl-2 family members

This study provides new insights into neuroprotection involving interaction of protein kinase C (PKC) pathway with Bcl‐2 family proteins. Using a model of serum deprivation, we investigated the mechanism by which the anti‐Parkinson/monoamine oxidase (MAO)‐B inhibitor drug, rasagiline, exerts its neuroprotective effect in rat pheochromocytoma PC12 cells. Here, we report that rasagiline (0.1–10 µM) decreased apoptosis via multiple protection mechanisms, including the stimulation of PKC phosphorylation; up‐regulation of PKCα and PKCε mRNAs, induction of Bcl‐xL, Bcl‐w, and brain‐derived neurotrophic factor (BDNF) mRNAs; and down‐regulation of Bad and Bax mRNAs. Moreover, rasagiline inhibited the cleavage and activation of procaspase‐3 and poly (ADP‐ribose) polymerase (PARP), whereas the PKC inhibitor, GF109203X, reversed these actions. Similarly, rasagiline decreased serum‐free‐induced levels of the important regulator of cell death, Bad, which was also blocked by GF109203X, indicating the involvement of PKC in rasagiline‐induced cell survival. Furthermore, these studies have established that PKC‐ and Bcl‐2‐dependent neuroprotective activity of rasagiline is dependent on its propargyl moiety, because propargylamine had similar effects with the same potency.

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition

Iron‐dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)‐B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinsons disease, Alzheimers disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO‐B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO‐B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO‐B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron‐dependent lipid peroxidation in rat brain homogenates with IC50 values (12–16 µm), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 µm were able to attenuate cell death induced by serum deprivation and by 6‐hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti‐Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non‐selective MAO‐A and MAO‐B inhibitor (IC50 < 0.1 µm). However, HLA20 was more selective for MAO‐B but had poor MAO inhibition, with an IC50 value of 64.2 µm. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

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: A novel anti-Parkinsonian monoamine oxidase-B inhibitor with neuroprotective activity

Rasagiline (N-propargyl-1-(R)-aminoindan) is a novel, highly potent irreversible monoamine oxidase (MAO)-B inhibitor, anti-Parkinsonian drug. Rasagiline is effective as monotherapy or adjunct to L-Dopa for patients with early and late Parkinsons disease (PD). Its S-isomer, TVP1022 is thousand times less potent as an MAO-B inhibitor. However, both compounds have similar molecular mechanisms of neuroprotection in neuronal cell cultures and animal neurodegenerative models, indicating that the neuroprotective effect of rasagiline does not depend on inhibition of MAO-B, but rather is associated with the N-propargyl moiety, which promotes mitochondrial viability and stabilizes permeability transition by regulating Bcl-2 family proteins. Novel findings demonstrated that the major metabolite of rasagiline, 1-(R)-aminoindan has antioxidant and neuroprotective capabilities and thus, may contribute to the overt activity of its parent compound, rasagiline. This paper will review the earlier and present studies in the development of rasagiline for treatment of PD and discuss its pharmacology and applicable mechanism of action.

The importance of propargylamine moiety in the anti-Parkinson drug rasagiline and its derivatives in MAPK-dependent amyloid precursor protein processing

Rasagiline [N‐propargyl‐(1R)‐aminoindan] a highly potent selective irreversible monoamine oxidase (MAO)‐B inhibitor exerts neuroprotective and antiapoptotic effects against a variety of insults in cell cultures and in vivo and has finished its phase III clinical trials for Parkinson‘s disease. In the present study, we show that rasagiline (1 and 10 μM) significantly protected rat PC12 cells against β‐amyloid (Aβ1‐42) toxicity. In addition, rasagiline significantly increased (approximately threefold) the secretion of the nonamyloidogenic soluble form of the amyloid precursor protein (sAPPα) from SH‐SY5Y neuroblastoma and PC12 cells. The increase of sAPPα was dose‐dependent and was blocked by the hydroxamic acid‐based metalloprotease inhibitor Ro31‐9790 (100 μM), suggesting that the effect is mediated via α‐secretase activity. Rasagiline‐induced sAPPα release was significantly reduced by the inhibitors of protein kinase C (PKC), GF109203X, and ERK mitogen‐activated protein kinase (MAPK) PD98059. Moreover, rasagiline dose dependently (0.1−10 μM) increased the phosphorylation of p44 and p42 MAPK, which was abolished by PD98059 (30 μM) and GF109203X (2.5 μM). By comparing the actions of rasagiline with those of its S‐isomer TVP1022, which is not an MAO inhibitor, we have been able to demonstrate that MAO‐B inhibition is not a prerequisite for either sAPPα‐induced release or ERK phosphorylation. In addition, structure‐activity relationship among rasagiline‐related compounds suggests the crucial role of the propargyl moiety in these molecules, because propargylamine itself significantly induced the secretion of sAPPα and increased MAPK phosphorylation with similar potency to that of rasagiline and its derivatives.

Therapeutic targets and potential of the novel brain- permeable multifunctional iron chelator-monoamine oxidase inhibitor drug, M-30, for the treatment of Alzheimer's disease.

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple CNS targets. We have synthesized a multifunctional non‐toxic, brain permeable iron chelator drug, M‐30, possessing propargyl monoamine oxidase (MAO) inhibitory neuroprotective and iron‐chelating moieties, from our prototype iron chelator VK‐28. In the present study M‐30 was shown to possess a wide range of pharmacological activities, including pro‐survival neurorescue effects, induction of neuronal differentiation and regulation of amyloid precursor protein (APP) and β‐amyloid (Aβ) levels. M‐30 was found to decrease apoptosis of SH‐SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via reduction of the pro‐apoptotic proteins Bad and Bax, and inhibition of the apoptosis‐associated phosphorylated H2A.X protein (Ser 139) and caspase 3 activation. In addition, M‐30 induced the outgrowth of neurites, triggered cell cycle arrest in G0/G1 phase and enhanced the expression of growth associated protein‐43. Furthermore, M‐30 markedly reduced the levels of cellular APP and β‐C‐terminal fragment (β‐CTF) and the levels of the amyloidogenic Aβ peptide in the medium of SH‐SY5Y cells and Chinese hamster ovary cells stably transfected with the APP ‘Swedish’ mutation. Levels of the non‐amyloidogenic soluble APPα and α‐CTF in the medium and cell lysate respectively were coordinately increased. These properties, together with its brain selective MAO inhibitory and propargylamine‐ dependent neuroprotective effects, suggest that M‐30 might serve as an ideal drug for neurodegenerative disorders, such as Parkinsons and Alzheimers diseases, in which oxidative stress and iron dysregulation have been implicated.

Involvement of MAP kinase in the regulation of amyloid precursor protein processing by novel cholinesterase inhibitors derived from rasagiline

Two novel neuroprotective cholinesterase (ChE) inhibitors, TV3326, (N‐propargyl‐(3R) aminoindan‐5‐yl)‐ethyl methyl carbamate, and TV3279, (N‐propargyl‐(3S) aminoindan‐5‐yl)ethyl methyl carbamate, were derived from rasagiline for the treatment of Alzheimers disease (AD). TV3326 also inhibits monoamine oxidase (MAO)‐A and ‐B, whereas its S‐isomer, TV3279, lacks MAO inhibitory activity. The action of these drugs in the regulation of amyloid precursor protein (APP) processing, using rat PC12 and human SH‐SY5Y neuroblastoma cells, was examined. Both isomers stimulated the release of the non‐amyloidogenic α‐secretase form of soluble APP (sAPPα) from these cell lines. The increases in sAPPα, induced by TV3326 and TV3279, were dose‐dependent (0.1–100 μM) and blocked by the hydroxamic acid‐based metalloprotease inhibitor, Ro31–9790, suggesting mediation via α‐secretase activity. Using several signal transduction inhibitors, we identified the involvement of protein kinase C (PKC), mitogen‐activated protein (MAP) kinase, and tyrosine kinase‐dependent pathways in the enhancement of sAPPα release by TV3326 and TV3279. In addition, both drugs directly induced the phosphorylation of p44 and p42 MAP kinase, which was abolished by the specific inhibitors of MAP kinase activation, PD98059 and U0126. These data suggest a novel pharmacological mechanism whereby these ChE inhibitors regulate the secretory processes of APP via activation of the MAP kinase pathway.

Regulation of protein kinase C by the anti-Parkinson drug, MAO-B inhibitor, rasagiline and its derivatives, in vivo

We have recently shown that the anti‐Parkinson‐propargyl‐containing monoamine oxidase B (MAO‐B) inhibitor drug, rasagiline [N‐propargyl‐(1R)‐aminoindan], and its cholinesterase inhibitor derivatives TV3326 and TV3279, regulate amyloid precursor protein (APP) processing by a protein kinase C (PKC)‐dependent mechanism in SH‐SY5Y neuroblastoma and PC12 cells. In the present study, we investigated the effect of rasagiline and its derivatives on the regulation of the PKC‐dependent mechanism and APP processing under in vivo conditions. Administration of rasagiline (0.1 mg/kg) to male C57/BL mice for 14 days significantly decreased membrane‐bound holoprotein APP levels in the hippocampus. Additionally, we observed that rasagiline up‐regulated p‐PKC levels and the expression of α and ε PKC isozymes in the hippocampus, indicating that the mechanism by which rasagiline affects APP processing may be related to PKC‐associated signalling. The results also demonstrate that rasagiline treatment significantly elevated the levels of phosphorylated myristoylated alanine‐rich C kinase substrate (p‐MARCKS), a major substrate for PKC, as well as the levels of receptors for activated C kinase 1 (RACK1). Similar effects on APP and PKC levels were also demonstrated for the two cholinesterase inhibitor derivatives of rasagiline, TV3326 and TV3279. These results indicate that rasagiline and its derivatives regulate PKC‐dependent mechanisms and APP processing. The activation and induction of PKC and MARCKS by these drugs may have a crucial role not only in their neuroprotective activity, but also in their ability to affect neuronal plasticity and spatial learning processes.

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.

Metallocorroles as cytoprotective agents against oxidative and nitrative stress in cellular models of neurodegeneration

J. Neurochem. (2010) 113, 363–373.