Neuroprotection by propargylamines in Parkinson's disease: intracellular mechanism underlying the anti-apoptotic function and search for clinical markers.

In Parkinson's and other neurodegenerative diseases, a therapeutic strategy has been proposed to halt progressive cell death. Propargylamine derivatives, rasagiline and (-)deprenyl (selegiline), have been confirmed to protect neurons against cell death induced by various insults in cellular and animal models of neurodegenerative disorders. In this paper, the mechanism and the markers of the neuroprotection are reviewed. Propargylamines prevent the mitochondrial permeabilization, membrane potential decline, cytochrome c release, caspase activation and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase. At the same time, rasagiline induces anti-apoptotic pro-survival proteins, Bcl-2 and glial cell-line derived neurotrophic factor, which is mediated by activated ERK-NF-kappaB signal pathway. DNA array studies indicate that rasagiline increases the expression of the genes coding mitochondrial energy synthesis, inhibitors of apoptosis, transcription factors, kinases and ubiquitin-proteasome system, sequentially in a time-dependent way. Products of cell survival-related gene induced by propargylamines may be applied as markers of neuroprotection in clinical samples.

The effect of neuromelanin on the proteasome activity in human dopaminergic SH-SY5Y cells.

In Parkinson's disease (PD), the selective depletion of dopamine neurons in the substantia nigra, particular those containing neuromelanin (NM), is the characteristic pathological feature. The role of NM in the cell death of dopamine neurons has been considered either to be neurotoxic or neuroprotective, but the precise mechanism has never been elucidated. In human brain, NM is synthesized by polymerization of dopamine and relating quinones, to which bind heavy metals including iron. The effects of NM prepared from human brain were examined using human dopaminergic SH-SY5Y cells. It was found that NM inhibits 26S proteasome activity through generation of reactive oxygen and nitrogen species from mitochondria. The mitochondrial dysfunction was also induced by oxidative stress mediated by iron released from NM. NM accumulated in dopamine neurons in ageing may determine the selective vulnerability of dopamine neurons in PD.

Involvement of type A monoamine oxidase in neurodegeneration: regulation of mitochondrial signaling leading to cell death or neuroprotection.

In neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, apoptosis is a common type of cell death, and mitochondria emerge as the major organelle to initiate death cascade. Monoamine oxidase (MAO) in the mitochondrial outer membrane produces hydrogen peroxide by oxidation of monoamine substrates, and induces oxidative stress resulting in neuronal degeneration. On the other hand, a series of inhibitors of type B MAO (MAO-B) protect neurons from cell death. These results suggest that MAO may be involved in the cell death process initiated in mitochondria. However, the direct involvement of MAO in the apoptotic signaling has been scarcely reported. In this paper, we present our recent results on the role of MAO in activating and regulating cell death processing in mitochondria. Type A MAO (MAO-A) was found to bind an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, and induce apoptosis in dopaminergic SH-SY5Y cells containing only MAO-A. To examine the intervention of MAO-B in apoptotic process, human MAO-B cDNA was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even though the activity and protein of MAO-B were expressed markedly. MAO-B oxidized dopamine with production of hydrogen peroxide, whereas in control cells expressing only MAO-A, dopamine autoxidation produced superoxide and dopamine-quinone, and induced mitochondrial permeability transition and apoptosis. Rasagiline and other MAO-B inhibitors prevent the activation of apoptotic cascade and induce prosurvival genes, such as bcl-2 and glial cell line-derived neurotrophic factor, in MAO-A-containing cells. These results demonstrate a novel function of MAO-A in the induction and regulation of apoptosis. Future studies will clarify more detailed mechanism behind regulation of mitochondrial death signaling by MAO-A, and bring out new strategies to cure or ameliorate the decline of neurons in neurodegenerative disorders.

Immobilized D-amino acid oxidase.

1. D-Amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) apoenzyme, holoenzyme and the enzyme-benzoate complex were active and stable when immobilized to aminoalkyl or carboxyalkyl agarose, or to cyanogen bromide-activated agarose. The immobilized enzyme-benzoate complex could be converted into the holo- and apoenzyme without being liberated from the agarose. 2. The apparent Michaelis constant and substrate specificity of the immobilized enzyme were similar to those of the free enzyme. The optimum pH of the reaction was shifted towards acidic side by 1.0-2.0 pH units from that of the free enzyme. 3. With increasing number of methylene groups of the 'spacer' from 3 to 5, molecular activity of the immobilized enzyme increased, while the apparent Miachaelis constant decreased.

Oxidation of sarcosine and N-alkyl derivatives of glycine by D-amino-acid oxidase.

1. Sarcosine was oxidized by D-amino-acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) to yield methylamine and glyoxylic acid. A seriies of N-alkyl glycines were also oxidized by this enzyme. 2. N-Acetyl- and N-Phenylglycine inhibited the oxidase by competing with the substrate, while N-methyl-N-acetylglycine did not bind to the enzyme. This suggests the requirement of at least one unsubstituted hydrogen atom at the amino group ofglycine for binding. 3. The primary step in the reaction was the release of a proton from the substrate, indicating the formation of a substituted imino acid, which was spontaneously hydrolyzed to glyoxylic acid acid and an amine.

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.

Branched-chain 2-oxo acid dehydrogenase complex activation by tetanic contractions in rat skeletal muscle.

Branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle was activated by muscle contractions elicited by electrical stimulation. This activation was attributed to dephosphorylation of the phosphorylated enzyme complex, and the total enzyme activity was not altered by muscle contractions. The activation of the enzyme complex occurred in the muscle of the electrically stimulated leg, but not in the muscle of the non-stimulated (control) leg, indicating that blood components are not involved in the mechanism of the enzyme activation in the muscle. Adenine nucleotides, branched-chain amino and 2-oxo acids and lactate in the muscle were determined as possible factors modulating the enzyme complex activity through inhibition of branched-chain 2-oxo acid dehydrogenase kinase activity. The profile of enzyme activation induced by muscle contractions was different from the alteration of the adenine nucleotide concentrations but was similar to the alteration of the concentrations of branched-chain amino and 2-oxo acids in the muscle. The lactate concentration in the stimulated muscle was elevated 3-5-fold during the contractions, indicating intracellular acidification. Previous studies have shown that the 2-oxo acid derived from leucine is a potent inhibitor of the kinase. These results suggest that intracellular branched-chain 2-oxo acids increased by muscle contractions accumulate in the mitochondria due to exercise-induced acidification of the muscle cell, resulting in activation of branched-chain 2-oxo acid dehydrogenase complex by inhibition of the kinase.

Purification and characterization of human liver beta-galactosidase from a patient with the adult form of GM1 gangliosidosis and a normal control.

beta-Galactosidases were purified to homogeneity from livers of a normal control and a patient with the adult form of GM1 gangliosidosis. The purification was achieved by chromatography on DEAE-Sepharose fast flow, Con A-Sepharose, p-aminophenyl-1-thio-beta-D-galactopyranoside-Sepharose, and QAE-Mono Q. The normal and mutant enzymes were purified about 5000-fold with a yield of 10% and 1800-fold with a yield of 34%, respectively, and could hydrolyze 4-methylumbelliferyl-beta-D-galactoside, GM1 ganglioside, and asialofetuin. The purified normal enzyme was eluted from a TSK gel G-4000SW column as three symmetrical peaks of protein which were coincident with the three peaks of enzyme activity. The enzyme in these three peaks had apparent molecular weights of 800,000 (polymer), 140,000 (dimer), and 65,000 (monomer), whereas the mutant enzyme was eluted as two symmetrical peaks of protein and enzyme activity. The apparent molecular weight of a major monomeric form of the enzyme (beta-galactosidase A) was 60,000, and no dimeric form of the enzyme existed. Normal and mutant purified enzyme preparations migrated as a single major protein band with apparent molecular weights of 65,000 or 60,000, respectively, by SDS-polyacrylamide gel electrophoresis after treatment with mercaptoethanol. On isoelectric focussing, the mutant enzyme migrated more anodally than the normal enzyme. The mutant enzyme also had altered enzyme properties, such as pH optimum, Km values, substrate specificity and heat-stability. These data on the characteristics of the purified enzyme preparations provide the first direct evidence that patients with the adult form of GM1 gangliosidosis have a structurally altered beta-galactosidase.

N-methyl(R)salsolinol produces hydroxyl radicals: involvement to neurotoxicity.

Recently, (R)-1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl-(R)salsolinol, NM(R)Sal] and 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion [DiMeDHIQ+] were found to cause a syndrome similar to parkinsonism in rodents. NM(R)Sal is produced in the brain by N-methylation of a naturally occurring catechol isoquinoline, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)salsolinol, (R)Sal], which is formed from dopamine. The mechanism of NM(R)Sal cytotoxicity to dopamine neurons was examined using in vitro experiments. NM(R)Sal was found to be nonenzymatically oxidized into DiMeDHIQ+, with concomitant formation of hydroxyl radicals. The oxidation and the radical production were completely inhibited by the antioxidants, ascorbic acid and reduced glutathione, and the radical formation was enhanced by Fe(II) and, to a less extent, by Fe(III). The oxidation of NM(R)Sal into DiMeDHIQ+ and the production of hydroxyl radicals may be essential for neurotoxicity to develop in dopamine neurons. The possible involvement of this catechol isoquinoline in the pathogenesis of Parkinson's disease is discussed.

Immunohistochemical detection of dityrosine in lipofuscin pigments in the aged human brain.

Lipofuscin is a yellowish brown fluorescent pigment which is sequestered within cytoplasmic granules during aging. To examine the contribution of protein oxidation to lipofuscin accumulation, we performed immunohistochemical detection of dityrosine, which is considered one of the specific markers for protein oxidation, in lipofuscin in the aged human brain using an antibody specific to dityrosine. By characterization using competitive enzyme-linked immunosorbent assay, the specificity of the antibody to dityrosine was confirmed. None of the other tyrosine-related compounds such as L-tyrosine, 3-nitrotyrosine, 3-chlorotyrosine, or 3,4-dihydroxyphenylalanine cross-reacted with the antibody. The anti-dityrosine antibody reacted with lipofuscin granules in the pyramidal neurons of the aged human brain. The results suggest that protein oxidation by free radicals and/or peroxidases may play an important role in lipofuscin accumulation.

Severe orthostatic hypotension in a female carrier of Fabry's disease.

A 21-year-old woman in a family with a history of Fabry's disease showed orthostatic hypotension and whorl-like corneal opacity typical for Fabry's disease. Biochemical studies revealed that she was a heterozygote of the Fabry gene. A variety of autonomic function tests demonstrated both sympathetic and parasympathetic dysfunction. To our knowledge, the present case is the first report of a heterozygous female carrier of Fabry's disease presenting dysfunction of the autonomic nervous system.

Cell death of dopamine neurons in aging and Parkinson's disease.

Dopamine neurons in the substantia nigra of human brain are selectively vulnerable and the number decline by aging at 5-10% per decade. Enzymatic and non-enzymatic oxidation of dopamine generates reactive oxygen species, which induces apoptotic cell death in dopamine neurons. Parkinson's disease (PD) is also caused by selective cell death of dopamine neurons in this brain region. The pathogenesis of Parkinson's disease remains to be an enigma, but it was found that an endogenous MPTP-like neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl(R)salsolinol, NM(R)Sal], may be one of the pathogenic agents of PD. NM(R)Sal increases in cerebrospinal fluid from untreated parkinsonian patients, and two enzymes, a (R)salsolinol synthase and a neutral N-methyltransferase, synthesize this neurotoxin in the nigro-striatum. The activity of a neutral N-methyltransferase is significantly higher in lymphocytes from parkinsonian patients than in control. The mechanism of cell death by this toxin was proved to be by the induction of apoptosis, by use of dopaminergic SH-SY5Y cells. The apoptosis was suppressed by anti-oxidants, suggesting that the generation of reactive oxygen species may initiate cellular death process. These results indicate that in aging and PD oxidative stress induces degeneration of dopamine neurons, and the antioxidant therapy may delay the decline of dopamine neurons in the brain.

Neuroprotection by (-)-deprenyl and related compounds.

There is an increasing number of data by in vitro and in vivo experiments, indicating that (-)-deprenyl is neuroprotective to dopamine neurons, even though detailed mechanism remains to be clarified. In this paper neuroprotection by (-)-deprenyl and structurally related compounds was examined in concern with the suppression of apoptosis induced by a reactive oxygen species, peroxynitrite generated from SIN-1. The apoptotic DNA damage was quantitatively determined using dopaminergic SH-SYSY cells and by a single cell gel electrophoresis (comet) assay. DNA damage induced by peroxynitrite was proved to be apoptotic by prevention of the damage by cycloheximide or actinomycin-D. (-)-Deprenyl and other propargylamines protected the cells from apoptosis in a dose-dependent way. (-)-Deprenyl protected the cells even after it was washed out, suggesting that it may initiate the intracellular process to repress the apoptotic death program. The study on the structure-activity relationship of (-)-deprenyl analogues revealed that a N-propargyl residue with adequate size of hydrophobic structure is essentially required for the anti-apoptotic activity. These results suggest that (-)-deprenyl and related compounds may protect neurons from apoptosis and be applicable to delay the deterioration of neurons during advancing ageing and in neurodegenerative disorders.

Mechanism underlying anti-apoptotic activity of a (-)deprenyl-related propargylamine, rasagiline.

A potent inhibitor of type B monoamine oxidase, (-)deprenyl, is known to protect or rescue dying neurons, independent of inhibition of the enzyme activity. After long term administration to rodents, a propargylamine structurally related to (-)deprenyl, (R)(+)-N-propargyl-1-aminoindan (rasagiline) increased the activities of anti-oxidative enzymes, superoxide dismutase and catalase. Rasagiline protected in vitro dopamine cells from apoptosis induced by oxidative stress or neurotoxins. The mechanism of the anti-apoptotic effect was studied by in vitro experiments using human dopaminergic neuroblastoma, SH-SY5Y cells. Peroxynitrite-generating N-morpholino sydonimine (SIN-1) induced apoptosis in SH-SY5Y cells via disruption of mitochondrial membrane potential (DeltaPsim), followed by caspase 3 activation. Rasagiline prevented the loss of DeltaPsim, the initial step to apoptosis, and also following caspase 3-activation and DNA fragmentation. The results suggest that rasagiline may interact with the specific molecule in the mitochondria and suppress the death signal transduction. By the anti-apoptotic function, rasagiline may rescue or protect declining neurons in aging and neurodegenerative disorders, such as Parkinson's disease.

A family with beta-galactosidase deficiency: three adults with atypical clinical patterns.

Three adult patients in a single family showed severe myoclonus, ataxia, and pyramidal signs. Enzymatic analysis of lymphocytes, plasma, and cultured skin fibroblasts showed marked deficiency of beta-galactosidase activity, more profound with GM1 ganglioside than with another natural substrate, asialofetuin. Other lysosomal hydrolases were normal. Although the physical signs were similar to those of types 1 and 2 GM1 gangliosidosis, none had bony abnormalities.

Atypical adult GM1 gangliosidosis: biochemical comparison with other forms of primary beta-galactosidase deficiency.

We studied beta-galactosidase in skin fibroblasts from patients with different forms of beta-galactosidase deficiency: adult GM1 gangliosidosis, type 1 GM1 gangliosidosis, and Morquio B syndrome. Enzyme properties in the adult cases differed from the other disorders and also from normal controls. Genetic hybridization studies indicated that all three forms belong to the same complementation group. Therefore, the adult disorder must be due to a mutation of the structural gene for beta-galactosidase, which is allelic to the mutations in type 1 GM1 gangliosidosis and Morquio B syndrome.

Inhibition of tryptophan hydroxylase by (R)- and (S)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines (salsolinols).

The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on PTH. These data indicate that salsolinols might be naturally occurring inhibitors of indoleamine metabolism.

Pancreas cancer associated antigen from normal colonic mucosa (PCAAc) assayed with monoclonal antibody.

We prepared monoclonal antibodies against pancreatic cancer associated antigen (PCAA) from normal colonic mucosa (PCAAc) and prepared an assay system to detect the circulating PCAAc in sera. 200 patients with cancer including 85 pancreatic cancer and benign disease were compared with 40 normal healthy individuals. Diagnostic rate of pancreatic cancer was 64/85 (75%) but other malignancy also showed elevated PCAAc: 18/19 (94.7%) of hepatoma and 19/37 (51.4%) of gastric cancer. In pancreatitis, 8/17 (47.1%) showed elevated PCAAc. Compared with our previous reports of PCAA assay, these results indicated that diagnostic sensitivity of pancreatic cancer was increased but specificity of it was decreased.

Inhibition of tryptophan hydroxylase by food-derived carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole.

Food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), were found to inhibit the activity of tryptophan hydroxylase (TPH) prepared from serotonin-producing murine mastocytomas, P-815 cells. Inhibition of TPH by Trp-P-2 was found to be competitive with the substrate L-tryptophan and non-competitive with the cofactor (6R)-L-erythro-5,6,7, 8-tetrahydrobiopterin. The inhibition proved to be reversible; by dialyzing the sample incubated with Trp-P-2, the enzyme activity could be fully recovered. Among a series of heterocyclic amines examined, Trp-P-1, Trp-P-2 and some other heterocyclic amines inhibited TPH activity. Trp-P-2 and other heterocyclic amines were the newly discovered naturally occurring inhibitors of the indoleamine metabolism.

Inhibition of tryptophan hydroxylase by dopamine and the precursor amino acids.

Effects of dopamine and its precursor amino acids on the activity of tryptophan hydroxylase were examined. They inhibited the enzyme activity prepared from mastocytoma cells in terms of the biopterin cofactor and the substrate L-tryptophan. In relation to the biopterin, tryptophan hydroxylase was found to have two different kinetics, and dopamine inhibited the activity in a non-competitive way to both the components. Dopamine had the highest affinity to the enzyme, followed by L-DOPA and L-tyrosine, while D-tyrosine did not inhibit the activity. In terms of L-tryptophan, L-tyrosine, L-DOPA and dopamine inhibited the enzyme non-competitively and their affinity to the enzyme was in this order. These results indicate that the indoleamine metabolism may be regulated by catecholamines and their related amino acids in the brain.