Anti-apoptotic function of propargylamine inhibitors of type-B monoamine oxidase.

In Parkinson's disease and other neurodegenerative diseases, (-)deprenyl, an inhibitor of type B monoamine oxidase (MAO-B), has been proposed to protect or rescue declining neurons. However, clinical trials failed to confirm the neuroprotection, even though in vivo and in vitro studies suggested the possibilities. This paper describes the activities of propargylamine MAO-B inhibitors against apoptosis induced by an endogenous selective dopaminergic neurotoxin, N-methyl(R)salsolinol, in dopaminergic SH-SY5Y cells. A series of propargylamines were shown to suppress the apoptotic cascade; preventing collapse of mitochondrial membrane potential, activation of caspase 3 and fragmentation of nucleosomal DNA. Among propargylamines, (R)-N-propargyl-1-aminoindan (rasagiline) was the most potent at preventing cell death. Rasagiline also prevented opening of permeability transition pore in insolated mitochondria. These results suggest that rasagiline and other propargylamines may regulate the apoptotic machinery in mitochondria and rescue or protect deteriorated neurons in neurodegenerative disorders.

Aliphatic propargylamines as symptomatic and neuroprotective treatments for neurodegenerative diseases.

Over the past several years, we have developed a number of novel aliphatic propargylamine-related compounds. These can be divided into 14 main chemical families. These families have been shown to possess members that selectively and stereochemically (i.e. R-enantiomer) rescue neurons from p53-dependent apoptosis in vitro. In contrast, no rescue has been observed by the enantiomers of the opposite configuration or in p53-independent apoptosis. In vivo, several compounds have been shown to possess neural rescue properties in models of unilateral hypoxia/ischaemia, focal ischaemia, facial nerve axotomy, pmn mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse and MPTP non-human primate. Our prototype compound, R-2HMP, has been shown to be metabolised in a manner analogous to that of R-deprenyl but devoid of amphetaminergic metabolites. These compounds have been shown to be active through an interaction with the same binding site as R-deprenyl and CGP 3466. This site is suggested to be the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Schizophrenia, a neurodegenerative disorder with neurodevelopmental antecedents.

Schizophrenia is a devastating disorder that has been referred to as youth's greatest disabler. Although a number of hypotheses have been proposed in an attempt to explain the pathophysiology of schizophrenia no single theory seems to account for all facets of the disease. Each hypothesis explains some of the phenomena associated with schizophrenia and it is probable that many variables described in these hypotheses interact to produce a disorder characterized by heterogeneous symptomatology, progression and prognosis. Compelling evidence suggests that the primary disturbance is a neurodevelopmental abnormality, possibly resulting from a genetic defect(s), resulting in a predisposition to schizophrenia. Events later in life may then lead to the presentation of symptoms and a subsequent progression of the disease. Recent evidence suggests that the progressive course of schizophrenia is associated with ongoing neurodegenerative processes. Changes in brain derived neurotrophic factor (BDNF) may explain the various changes observed in schizophrenia.

Enantio-specific induction of apoptosis by an endogenous neurotoxin, N-methyl(R)salsolinol, in dopaminergic SH-SY5Y cells: suppression of apoptosis by N-(2-heptyl)-N-methylpropargylamine.

Endogenous N-methyl(R)salsolinol, which caused parkinsonism in rats by injection in the striatum, was found to induce apoptosis in dopaminergic neuroblastoma SH-SY5Y cells. After 12-h incubation with 500[microM N-methyl(R)salsolinol, almost all the cells died with apoptosis and necrotic cell death was negligible. N-Methyl(R)salsolinol was much more potent to induce apoptosis than the (S)-enantiomer. The mechanism of apoptosis was studied in relation to changes in mitochondrial membrane potential, deltapsi(m), using a fluorescent indicator, JC-1. Red fluorescence of J-aggregates representing hyperpolarized deltapsi(m) was found to decrease significantly within 60 min after incubation with N-methyl(R)salsolinol, but not by the (S)-enantiomer at the same concentration. It suggests that mitochondria may recognize the stereo-chemical structure of N-methyl(R) salsolinol. Aliphatic propargylamines, (R)-N-(2-heptyl)-N-methylpropargylamine and (R)-N-(2-heptyl)propargylamine, were found to prevent deltapsim loss and subsequent apoptosis induced by N-methyl(R)salsolinol. These results suggest that mitochondria play a key role in the induction of apoptosis by the neurotoxin and the prevention by aliphatic propargylamines.

Glyceraldehyde-3-phosphate dehydrogenase and apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as playing an integral role in glycolysis. During the past 20 years, however, a number of novel, additional functions for GAPDH have been described. These include acting as an uracil DNA glycosylase, activator of transcription, binding to RNA, and an involvement in tubulin assembly. One of the most intriguing functions which has recently been recognized is an involvement in the initiation of apoptosis. Further, GAPDH associates with proteins implicated in human neurodegenerative disorders. This review summarizes the evidence implicating GAPDH in the initiation of one or more apoptotic cascades. The possible functions of GAPDH in the nucleus which could result in the initiation of apoptosis are also discussed.

Metabolism and pharmacokinetics, in the rat, of (R)-N-(2-Heptyl)Methyl-propargylamine (R-2HMP), a new potent monoamine oxidase inhibitor and antiapoptotic agent.

(R)-N-(2-Heptyl)-N-methylpropargylamine (R-2HMP) is a monoamine oxidase inhibitor and putative antiapoptotic agent analogous to (R)-deprenyl. In the rat, the major amine metabolites of R-2HMP have been identified as (R)-N-2-heptylmethylamine (R-2HMA), (R)-N-2-heptylpropargylamine (R-2HPA), and (R)-2-heptylamine (R-2HA). After R-2HMP was administered s.c. to male Wistar rats, it was observed that the greatest concentration was of the original drug followed in decreasing order by R-2HMA, R-2HPA, and R-2HA in brain, liver, and plasma at all times after administration. The greatest concentrations of the three metabolites were found in brain followed by liver and plasma, and the peak concentrations occurred between 15 and 30 min after administration. After oral administration, the liver contained the greatest concentrations of drug and metabolites, and, again, the peak concentrations occurred at about 15 min. In all cases, depropargylation appears to occur at a faster rate than demethylation. After s.c. administration, R-2HMP and its metabolites exhibited biexponential redistribution and elimination losses. Half-lives of the compounds in brain for the redistribution phase were: R-2HMP, 10 min; R-2HMA, 11 min; R-2HPA, 16 min; and R-2HA, 15 min.

Neurorescuing effects of the GAPDH ligand CGP 3466B.

(-)-Deprenyl, used for the treatment of Parkinson's disease, was reported to possess neurorescuing/antiapoptotic effects independent of its MAO-B inhibiting properties. It is metabolized to (-)-desmethyldeprenyl, which seems to be the active principle, and further to (-)-amphetamine and (-)-methamphetamine, which antagonize its rescuing effects. These complications may explain the limited neurorescuing potential of (-)-deprenyl observed clinically. CGP 3466 (dibenzo[b,f]oxepin-10-ylmethyl-methyl-prop-2-ynyl-amine), structurally related to (-)-deprenyl, exhibits virtually no MAO-B nor MAO-A inhibiting properties and is not metabolized to amphetamines. It was shown to bind to glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme with multiple other functions including an involvement in apoptosis, and shows neurorescuing properties qualitatively similar to, but about 100-fold more potent than those of (-)-deprenyl in several in vitro and in vivo paradigms. In concentrations ranging from 10(-13)-10(-5) M, it rescues partially differentiated PC12 cells from apoptosis induced by trophic withdrawal, cerebellar granule cells from apoptosis induced by cytosine arabinoside, rat embryonic mesencephalic dopaminergic cells from death caused by MPP+, and PAJU human neuroblastoma cells from death caused by rotenone. However, it did not affect apoptosis elicited by a variety of agents in rapidly proliferating cells from thymus or skin or in liver or kidney cells. In vivo, it rescued facial motor neuron cell bodies in rat pups after axotomy, rat hippocampal CA1 neurons after transient ischemia/hypoxia, and mouse nigral dopaminergic cell bodies from death induced by MPTP, in doses ranging between 0.0003 and 0.1 mg/kg p.o. or s.c., depending on the model. It also partially prevented the loss of tyrosine hydroxylase immunoreactivity in the substantia nigra of 6-OHDA-lesioned rats and improved motor function in these animals. Moreover, it prolonged the life-span of progressive motor neuronopathy (pmn) mice (a model for ALS), preserved their body weight and improved their motor performance. This was accompanied by a decreased loss of motor neurons and motor neuron fibers, and protection of mitochondria. The active concentration- or dose-ranges in the different in vitro and in vivo paradigms were remarkably similar. In several paradigms, bell-shaped dose-response curves were observed, the rescuing effect being lost above about 1 mg/kg, a fact that must be considered in clinical investigations.

Loss of mitochondrial membrane potential is dependent on the apoptotic program activated: prevention by R-2HMP.

Recent evidence suggests that the mitochondrial membrane potential begins to decrease well before the cells commit to apoptotic death. By using cultured cerebellar granule cells, two types of apoptosis can be induced, one by adding cytosine arabinoside (Ara-c; p53-dependent apoptosis) and one by lowering the K(+) concentrations of the medium (p53-independent apoptosis). Cultures show clear signs of increased apoptosis (chromatin condensation as visualized with bis-benzamide) after 12 hr which increases with time up to 24 hr. A fluorescent probe, chloromethyl-tetramethylrhodamine methyl ester (CMTMR), a lipophilic, potentiometric dye, which when introduced into the media accumulates within mitochondria in proportion to the mitochondrial membrane potential, was added at various time points after the induction of apoptosis. In Ara-c-induced apoptosis, there was a shift in the distribution of cell populations towards low-intensity CMTMR fluorescence, whereas in control and low-K(+) cultures, there was no such shift. This effect was observed as early as 6 hr after adding Ara-c. The antiapoptotic drug R-N-2-heptyl-N-methylpropargylamine hydrochloride (R-2HMP) reversed this loss of mitochondrial membrane potential in Ara-c-induced apoptosis; the effect was antagonized by the S-2HMP.

MK-801-induced expression of Fos protein family members in the rat retrosplenial granular cortex.

The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 has been shown to induce an acute Fos and Fos-related antigen (Fra) expression in the rat retrosplenial granular cortex (RSG), but the exact composition of the Fos protein family and their individual dynamic alterations are unknown. We examined this issue using immunocytochemistry and Western blot analysis with two antibodies that recognize, respectively, Fos and all the identified members of Fos protein family. Immunocytochemistry detected a rapid and transient expression of Fos proteins in some RSG neurons and a delayed and prolonged expression of Fra proteins in most RSG neurons following a single systemic MK-801 injection (1 mg/kg). Multiple MK-801 injections (i.e., ten consecutive injections once every other day; 1 mg/kg) produced a moderate Fra expression but failed to induce detectable Fos expression. Western blot analysis further showed a transient expression of 72-kDa Fos proteins following a single MK-801 injection and a prolonged expression of 46- and 43-kDa Fra proteins after either a single or multiple MK-801 administration. The delayed onset and prolonged expression of these Fra proteins suggest that they may be DeltaFosB. The possible relevance of these results to clinical psychotomimetic effects of the NMDA receptor antagonists phencyclidine and ketamine is discussed.

Differential effects of olanzapine on the gene expression of superoxide dismutase and the low affinity nerve growth factor receptor.

Neuroanatomical studies of schizophrenia suggest that progressive neuropathological changes (such as neuronal atrophy and/or cell death) occur over the lifetime course of the disease. Early intervention with atypical neuroleptics has been shown to prevent progression of at least some symptoms, although the mechanisms by which neuroleptics may do this remain unknown. In this study, PC12 cells were used to determine the effects of the new atypical antipsychotic olanzapine on the gene expression of superoxide dismutase (SOD1) and the low affinity nerve growth factor receptor (p75). The results show that olanzapine increases SOD1 at concentrations of 10 and 100 microM after 48 hr of incubation in PC12 cultures. The treatment decreases p75 gene expression at concentrations 100 microM after 48 hr of incubation. Since both the upregulation of SOD1 mRNA and the antisense blockade of p75 mRNA have been associated with reduced cell death, our results suggest that olanzapine has neuroprotective potential and thus may be useful in preventing further neurodegeneration accompanying schizophrenia.

Prolongation of life in an experimental model of aging in Drosophila melanogaster.

(R)-Deprenyl, the archetypical monoamine oxidase-B inhibitor, has been shown to increase life-span in a number of species. Although many theories for this effect have been suggested, for example, an increase in superoxide dismutase (SOD) activity, the mechanism of action has yet to be elucidated. To investigate this phenomenon, we have examined the effects of (R)-deprenyl, and some aliphatic propargylamines, in an experimental aging model in Drosophila melanogaster. Both wild-type Oregon-R type flies, as well as a SOD knock-out mutant strain were used. Flies obtained from a series of paired mates were divided equally among treatment groups. In all studies, flies were treated for the duration of life following adult emergence. The aging model consists of substitution of sucrose with galactose in the regular food media of the flies. Initial experiments confirmed that such a substitution resulted in a significant (p < 0.01, Breslow test) reduction in mean and maximal life-span of flies, an effect not due to nutrient deprivation. Inclusion of (R)-deprenyl and the aliphatic propargylamines in the media, at average daily doses in the range 0.5-1 ng/fly/day, led to a significant increase in mean and maximal life-span of galactose-treated, but not control flies. This effect was seen in both wild-type and mutant flies.

Symptomatic and neuroprotective properties of the aliphatic propargylamines.

This short review describes the development of several families of aliphatic propargylamine drugs. The parent family--the aliphatic N-methylpropargylamines are potent, specific, irreversible MAO-B inhibitors, like R-deprenyl; but devoid of R-deprenyl's toxicity and amphetaminergic effects. In addition the parent drugs are potent antiapoptoic agents. Their desmethyl and further oxidized metabolites whilst devoid of MAO-B inhibitory properties of R-2HMP (our chosen drug candidate) are described along with the suggestion that this drug be developed for the treatment of several human neurodegenerative conditions.

Modeling and optimization of enantioseparation by capillary electrochromatography.

Both electrophoretic and chromatographic transport mechanisms are combined in electrochromatographic separation. In this paper, we developed a model of enantioselectivity in capillary electrochromatography (CEC) which can be applied in the separation of both neutral and ionic compounds. The overall selectivity in enantioseparation is considered to be made up of two contributions: one is the intrinsic difference in formation constants of a pair of enantiomers, and the other is the conversion efficiency of the intrinsic difference into the apparent difference in the migration velocity. The model was illustrated through the chiral separation of (R)- and (S)-salsolinols. Under a positive electric field, enantioseparation of salsolinols was achieved on an ODS column with beta-cyclodextrin as a chiral mobile-phase additive. The experimental results are discussed in relation to the effect of separation parameters, such as the direction and size of electric field and properties of the stationary and mobile phases. It was demonstrated that if both electrophoretic and partitioning mechanisms produce positive effects, high overall selectivity in CEC can be obtained. For pressurized-driven electrochromatography, although the column efficiency is sacrificed due to the introduction of hydrodynamic flow, the increased selectivity significantly reduced the requirement of large column plate numbers for resolution.

Amantadine increases aromatic L-amino acid decarboxylase mRNA in PC12 cells.

Amantadine is an antiviral agent that was unexpectedly found to cause symptomatic improvement in patients with Parkinsonism, although its mechanism of action remains to be elucidated. Aromatic L-amino acid decarboxylase (AADC) is a regulated enzyme that catalyzes the decarboxylation of 3,4-dihydroxyphenylalanine (L-Dopa). It may be especially important during L-Dopa therapy in Parkinsonism, during which it may be rate-limiting for the production of dopamine. This study reports the effects of amantadine on the gene expression of AADC in PC12 cells. It shows that amantadine induces AADC gene expression at concentrations of 10 and 100 microM after 24 hr of incubation. The results suggest that the stimulation of AADC mRNA by amantadine may be one of its effects on dopamine metabolism that may have relevance for potentiation of L-Dopa therapy in Parkinsonism.

L-deprenyl potentiates NGF-induced changes in superoxide dismutase mRNA in PC12 cells.

L-deprenyl protects neurons in a number of in vivo and in vitro models and it has been postulated that it ameliorates some neurodegenerative disorders. Superoxide dismutase (SOD) is one of the enzymes responsible for the inactivation of oxygen free radicals, and one of the mechanisms of the effect of L-deprenyl is thought to act by induction of SOD. In this study, PC12 cells were used to study the effect of L-deprenyl on gene regulation of SOD and its interaction with nerve growth factor (NGF). The results show that NGF induces SOD mRNA in a dose-dependent manner and that a similar effect was produced by L-deprenyl. In addition, L-deprenyl potentiates NGF effects. This study demonstrates that both L-deprenyl and NGF may be involved in common antioxidative mechanisms and that L-deprenyl may interact with neurotrophic factors.

R-deprenyl and R-2-heptyl-N-methylpropargylamine prevent apoptosis in cerebellar granule neurons induced by cytosine arabinoside but not low extracellular potassium.

R-Deprenyl and R-2-heptyl-N-methylpropargylamine (R-2-HMP) are compounds that have been shown to reduce neuronal death in various in vitro and in vivo models involving apoptosis but do not always prevent apoptosis. In the present study we have examined the effects of these compounds and their S enantiomers on cytosine arabinoside (ara C)-induced apoptosis and low K+-induced apoptosis in cerebellar granule cells in primary culture. It was found that R-deprenyl and R-2-HMP could prevent ara C-induced apoptosis with an EC50 around 10(-9) M but could not prevent low K+-induced apoptosis. S-Deprenyl and S-2-HMP did not prevent apoptosis under any conditions but were found to antagonize the antiapoptotic actions of R-deprenyl and R-2-HMP. Using the fluorescent mitochondrial dye chloromethyltetramethylrhodamine methyl ester it was found that there was a loss of mitochondrial function in cerebellar granule cells exposed to ara C but not low K + medium. R-Deprenyl and R-2-HMP prevented the ara C-induced loss of mitochondrial function. It is concluded that R-deprenyl and R-2-HMP prevent apoptosis of cerebellar granule cells by a mechanism that is independent of monoamine oxidase inhibition and that they act on the same site to prevent specifically apoptosis involving a loss of mitochondrial membrane potential, possibly p53-dependent apoptosis.

L-deprenyl induces aromatic L-amino acid decarboxylase (AADC) mRNA in the rat substantia nigra and ventral tegmentum. An in situ hybridization study.

L-Deprenyl is a complex drug, and number of mechanisms have been proposed to explain its effects. These include blockade of dopamine metabolism, amplification of dopamine responses, induction of superoxide dismutase or delaying apoptosis. Using in situ hybridization techniques, we have shown that L-deprenyl (5-10 mg/kg intraperitoneally, killed after 24 h) increases aromatic L-amino acid decarboxylase (AADC) mRNA levels in rat substantia nigraventral tegmental area. In human brain tissue, AADC is present at low levels, suggesting a possible rate-limiting role in monoamine synthesis. This is particularly important in parkinsonian patients, since the therapeutic efficacy of L-DOPA is attributed to its enzymatic decarboxylation to dopamine. The present findings support that one of the effects of L-deprenyl may be to facilitate the decarboxylation of L-DOPA by increasing the availability of AADC.

Aliphatic N-methylpropargylamines as potential neurorescue agents.

Several clinical investigations have indicated that R-deprenyl, a typical monoamine oxidas B inhibitor, delays the progression of Parkinson's and Alzheimer's disease. A number of aliphatic N-methylpropargylamines, such as R-2-hexyl-N-methylpropargylamines (R-2HxMP), have been found to be highly potent, irreversible, selective, MAO-B inhibitors both in vitro and in vivo. These aliphatic propargylamines do not affect noradrenaline of dopamine uptake and are chemically without an amphetamine moiety and therefore do not exhibit any amphetamine-like effects. They are capable of protecting mouse striatal dopamine neurons against MPTP-induced toxicity in the caudate, against MK-801-induced apoptosis in the retrosplenial cortex and against DSP-4-induced depletion of naradrenergic axons. They rescue hippocampal neurons in rodents following kainate-induced neuronal damage. They block the expression of heat shock protein (HSP70) and delayed c-Fos expression in hippocampal CA1 region as elicited by kainate. Confocal microscopy also revealed prevention of neuronal damage in hippocampal slices under hypoxia-hypoglycemia conditions. Aliphatic N-methylpropargylamines may be useful in the treatment of neurodegenerative disorders. The mechanism and site of action of the neurorescue effect of these propargylamines, however, remains to be established.