Merav Yogev-Falach

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.

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.

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.

A multifunctional, neuroprotective drug, ladostigil (TV3326), regulates holo-APP translation and processing

The recent therapeutic approach in which drug candidates are designed to possess diverse pharmacological properties and act on multiple targets has stimulated the development of the bifunctional drug ladostigil (TV3326) [(N‐propargyl‐(3R) aminoindan‐5yl)‐ethyl methyl carbamate]. Ladostigil combines the neuroprotective effects of the antiparkinson drug rasagiline, a selective monoamine oxidase (MAO)‐B inhibitor, with the cholinesterase (ChE) inhibitory activity of rivastigmine in a single molecule, as a potential treatment for Alzheimers disease (AD) and Lewy Body disease. Here, we assessed the dual effects of lodostigil in terms of the molecular mechanism of neuroprotection and amyloid precursor protein (APP) regulation/processing by using an apoptotic model of neuroblastoma SKN‐SH cells. Ladostigil dose‐dependently decreased cell death via inhibition of the cleavage and prevention of caspase‐3 activation (IC501.05 µM) through a mechanism related to regulation of the Bcl‐2 family proteins, which resulted in reduced levels of Bad and Bax and induced levels of Bcl‐2 gene and protein expression. We have also followed APP regulation/ processing and found that ladostigil markedly decreased apoptotic‐induced levels of holo‐APP protein without altering APP mRNA levels, suggesting a posttranscriptional mechanism. In addition, the drug‐elevated phosphorylated protein kinase C (pPKC) levels and stimulated the release of the nonamyloidogenic ‐secretase proteolytic pathway. Similar to ladostigil, its S‐isomer, TV3279, which is a ChE inhibitor but lacks MAO inhibitory activity, exerted neuroprotective properties and regulated APP processing, indicating that these effects are independent of MAO inhibition.—Yogev‐Falach, M., Bar‐Am, O., Amit, T., Weinreb, O., Youdim, M. B. H. The multifunctional neuroprotective anti‐Alzheimer/ anti‐Parkinson drug ladostigil (TV3326) regulates holo‐APP translation and processing. FASEB J. 20, E1610 –E1618 (2006)

Amyloid Processing and Signal Transduction Properties of Antiparkinson‐Antialzheimer Neuroprotective Drugs Rasagiline and TV3326

Abstract: Two novel neuroprotective cholinesterase (ChE) inhibitors, TV3326 and TV3279 [(N‐propargyl‐(3R) and (3S) aminoindan‐5‐yl)‐ethyl methyl carbamate], respectively were derived from rasagiline, for the treatment of Alzheimers disease (AD). TV3326 also inhibits monoamine oxidase (MAO)‐A and B, while its S‐isomer, TV3279, lacks MAO‐inhibitory activity. The actions of these drugs in the regulation of the amyloid precursor protein (APP) processing using rat PC12 and human SH‐SY5Y neuroblastoma cells were 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, 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 was identified. 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 secretary processes of APP via activation of the MAP kinase pathway.

The neuroprotective mechanism of action of the multimodal drug ladostigil.

The recent therapeutic approach in which drug candidates are designed to possess diverse pharmacological properties and act on multiple targets has stimulated the development of the multimodal drug, ladostigil (TV3326) ((N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate). Ladostigil combines neuroprotective effects with monoamine oxidase -A and -B and cholinesterase inhibitory activities in a single molecule, as a potential treatment for Alzheimers disease (AD) and Lewy Body disease. Preclinical studies show that ladostigil has antidepressant and anti-AD activities and the clinical development is planned for these dementias. In this review, we discuss the multimodal effects of ladostigil in terms of neuroprotective molecular mechanism in vivo and in vitro, which include the amyloid precursor protein processing; activation of protein kinase C and mitogen-activated protein kinase pathways; regulation of the Bcl-2 family members; inhibition of cell death markers and up-regulation of neurotrophic factors. Altogether, these scientific findings make ladostigil a potentially valuable drug for the treatment of AD.

Molecular imaging of neurodegeneration by a novel cross-disease biomarker.

Current pre-mortem diagnosis of neurodegenerative disorders such as Alzheimers disease (AD) or amyotrophic lateral sclerosis (ALS) is based on clinical assessment of neurological deficits. However, symptoms and signs emerge only late in the disease course, thus indicating an urgent need for novel tools for detection of the underlying neuropathology. NST-729 (MW=310) is a novel molecular imaging probe, which is a member of the ApoSense family of small molecule detectors of apoptosis. We now report on the ability of NST-729, upon its systemic administration in vivo, to detect characteristic neuropathology in pre-clinical models of AD (Tg2576 transgenic mice) and ALS (transgenic SOD-1 G93A mutation mice). In the AD model, NST-729 clearly and selectively bound and imaged amyloid plaques, in excellent correlation with a typical amyloid ex vivo staining (Congo red). In the ALS model, NST-729 distinctly and selectively imaged multiple degenerating neurons in the motor nuclei in the pons, medulla and spinal cord, manifesting numerous multifocal irregularities and disruptions of neuritic projections, typical of axonal apoptosis. Study results therefore support the potential utility of NST-729 as a cross-disease biomarker for neurodegeneration, and also its potential role as the first molecular probe for ALS. Future radio-labeled NST-729 analogues may assist in the early diagnosis of disease, and in the development of neuroprotective therapies for these severe neurological disorders.