Neuroprotection by deprenyl and other propargylamines: glyceraldehyde-3-phosphate dehydrogenase rather than monoamine oxidase B.

Deprenyl and other propargylamines are clinically beneficial in Parkinson's disease (PD). The benefits were thought to depend on monoamine oxidase B (MAO-B) inhibition. A large body of research has now shown that the propargylamines increase neuronal survival independently of MAO-B inhibition by interfering with apoptosis signaling pathways. The propargylamines bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH binding is associated with decreased synthesis of pro-apoptotic proteins like BAX, c-JUN and GAPDH but increased synthesis of anti-apoptotic proteins like BCL-2, Cu-Zn superoxide dismutase and heat shock protein 70. Anti-apoptotic propargylamines that do not inhibit MAO-B are now in PD clinical trial.

Propargylamines induce antiapoptotic new protein synthesis in serum- and nerve growth factor (NGF)-withdrawn, NGF-differentiated PC-12 cells.

(-)-Deprenyl and structurally related propargylamines increase neuronal survival independently of monoamine oxidase B (MAO-B) inhibition, in part by decreasing apoptosis. We found that deprenyl and two other propargylamines, one of which does not inhibit monoamine oxidase B, increased survival in trophically withdrawn 6-day nerve growth factor (NGF)- and 9-day NGF-differentiated PC-12 cells but not in NGF naive or 3-day NGF-differentiated PC-12 cells. Four days of prior NGF exposure were required for the propargylamine-mediated antiapoptosis. Studies using actinomycin D, cycloheximide, and camptothecin revealed that the maintenance of both transcription and translation, particularly between 2 and 6 h after trophic withdrawal, was required for propargylamine-mediated antiapoptosis. Metabolic labeling of newly synthesized proteins for two-dimensional protein gel autoradiography and scintillation counting showed that the propargylamines either increased or reduced the levels of new synthesis or induced de novo synthesis of a number of different proteins, most notably proteins in the mitochondrial and nuclear subfractions. Western blotting for whole cell or subcellular fraction lysates showed that the timing of new protein synthesis changes or subcellular redistribution of apoptosis-related proteins induced by the propargylamines were appropriate to antiapoptosis. The apoptosis-related proteins included superoxide dismutases (SOD1 and SOD2), glutathione peroxidase, c-JUN, and glyceraldehyde-3-phosphate dehydrogenase. Most notable were the prevention of apoptotic decreases in BCL-2 levels and increases in mitochondrial BAX levels. In general, (-)-deprenyl-related propargylamines appear to reduce apoptosis by altering the levels or subcellular localization of proteins that affect mitochondrial membrane permeability, scavenge oxidative radicals, or participate in specific apoptosis signaling pathways.

Involvement of apoptosis in neurological injury after hypothermic circulatory arrest: a new target for therapeutic intervention?

BACKGROUND: This study was undertaken to evaluate the role of apoptosis in neurological injury after hypothermic circulatory arrest (HCA). METHODS: Twenty-one pigs (27 to 31 kg) underwent 90 minutes of HCA at 20 degrees C and were electively sacrificed at 6, 24, 48, and 72 hours, and at 7, 10, and 12 days after HCA, and compared with unoperated controls. In addition, 3 animals that had HCA at 10 degrees C, and 3 treated with cyclosporine A (CsA) in conjunction with HCA at 20 degrees C, were examined 72 hours after HCA. After selective perfusion and cryopreservation, all brains were examined to visualize apoptotic DNA fragmentation and chromatin condensation on the same cryosection of the hippocampus: fluorescent in situ end labeling (ISEL) was combined with staining with a nucleic acid-binding cyanine dye (YOYO). RESULTS: In addition to apoptosis, which was seen at a significantly higher level (p = 0.05) after HCA than in controls, two other characteristic degenerative morphological cell types (not seen in controls) were characterized after HCA. Cell death began 6 hours after HCA and reached its peak at 72 hours, but continued for at least 7 days. Compared with the standard protocol at 20 degrees C, HCA at 10 degrees C and CsA treatment both significantly reduced overall cell death after HCA, but not apoptosis. CONCLUSIONS: The data establish that significant neuronal apoptosis occurs as a consequence of HCA, but at 20 degrees C, other pathways of cell death, probably including necrosis, predominate. Although preliminary results suggest that the neuroprotective effects of lower temperature and of CsA are not a consequence of blockade of apoptotic pathways, inhibition of apoptosis nevertheless seems promising as a strategy to protect the brain from the subtle neurological injury that is associated with prolonged HCA at clinically relevant temperatures.

Apoptosis and anti-apoptosis signalling in glaucomatous retinopathy.

PURPOSE: Recent studies in the post-mortem human retina have suggested that apoptosis contributes to retinal ganglion cell (RGC) loss in glaucoma. If apoptosis contributes significantly to glaucomatous RGC loss, and if the specific apoptosis signalling pathways for glaucomatous apoptosis can be determined, agents that interrupt or oppose the signalling have the potential to slow the progression of glaucoma. METHODS: Recent data in animal models indicate that mitochondrially-dependent apoptosis contributes to RGC loss in glaucoma. Mitochondrially-dependent apoptosis involves proteins like BAX that increase mitochondrial membrane permeability and promote apoptosis and proteins like BCL-2 that decrease mitochondrial membrane permeability and reduce apoptosis. New protein synthesis induced by the alpha-2 agonist, brimonidine, prevents decreases in the levels of BCL-2 and thereby reduces mitochondrially-dependent apoptosis. CONCLUSIONS: Brimonidine appears to maintain BCL-2 levels by supporting the activity of an intrinsic anti-apoptosis signalling system that involves phosphorylation of protein kinase B. Phosphorylated protein kinase B appears to counteract the apoptosis signalling mechanisms which operate in glaucomatous retina.

Apoptotic cell death in the hippocampus due to prolonged hypothermic circulatory arrest: comparison of cyclosporine A and cycloheximide on neuron survival.

OBJECTIVE: To determine whether cyclosporine A (CsA) or cycloheximide (CHX) can reduce neuronal apoptosis in the hippocampus in a chronic animal model of hypothermic circulatory arrest (HCA). METHODS: Twenty-eight pigs (28-33 kg) underwent 90 min of HCA at 20 degrees C. In a blinded study, animals were randomized to placebo (n=12), 5 mg/kg CsA (n=8), or 1 mg/kg CHX (n=8). After elective sacrifice 7 days postoperatively, brains were perfusion-fixed and the left hippocampus was examined for evidence of neuronal cell death. An in situ double-labeling method was used on cryosections to unequivocally identify apoptotic nuclei by the simultaneous visualization of DNA fragmentation and apoptotic chromatin condensation. Sections were also examined by immunocytochemistry for upregulation of the pro-apoptotic proteins Bax, activated caspase 3, and glyceraldehyde-3-phosphate dehydrogenase. RESULTS: Apoptotic nuclear degradation was clearly present in the CA1, CA2 and CA3 subregions of the hippocampus after HCA. However, there was also morphological evidence for an accompanying necrotic-like cell death. There was no significant difference between the number of apoptotic nuclei observed in CSA-treated animals, mean value 4.4+/-1.63 SEM or CHX-treated animals, mean value 4.0+/-1.92 SEM, and age-matched control HCA pigs, mean value 4.85+/-1.69 SEM, (P>0.10). CONCLUSIONS: The data clearly demonstrate apoptotic cell death in pigs after HCA by simultaneously demonstrating in situ end labeling (TUNEL reaction) and apoptotic chromatin condensation using a nucleic acid-binding dye. Since CsA shows promising neuroprotective effects in behavioral studies, and since the peak of HCA-induced apoptosis occurs earlier than 7 days, further studies will be required to determine whether CsA can improve neuronal survival in the first few days after HCA. CHX was not effective in reducing apoptosis in this model.

Cyclosporine A as a potential neuroprotective agent: a study of prolonged hypothermic circulatory arrest in a chronic porcine model.

OBJECTIVE: To assess whether Cyclosporine A (CsA) or cycloheximide (CHX) can reduce ischemia-induced neurological damage by blocking apoptotic pathways, we assessed their effects on cerebral recovery in a chronic animal model of hypothermic circulatory arrest (HCA). METHODS: Twenty-eight pigs (28-33 kg) underwent 90 min of HCA at 20 degrees C. In this blinded study, animals were randomized to placebo (n=12), 5 mg/kg CsA (n=8), given intravenously before and subcutaneously for 7 days after HCA, or a single dose of 1 mg/kg CHX (n=8), given after weaning from cardiopulmonary bypass. Hemodynamics, intracranial pressure (ICP) and neurophysiological data (EEG, SSEP) were assessed for 3 h after HCA; early behavioral recovery was scored, and neurological/behavioral evaluation (9=normal) was carried out daily until elective sacrifice on postoperative day (POD) 7. Brains were selectively perfused and evaluated histopathologically for apoptosis. RESULTS: Basic hemodynamic data revealed no differences between CsA or CHX and control groups. ICP was significantly lower throughout rewarming (P=0.009) and reperfusion (P=0.05) in the CsA group. EEG recovery 3 h after HCA was observed in four of eight CsA animals but in only 1 of 12 controls (P=0.11) and one of eight CHX animals; cortical SSEP recovery also seemed faster in CsA animals, but failed to reach significance. Some early recovery scores were significantly better in the CsA group, and daily behavioral scores were consistently and significantly higher in the CsA-treated animals from POD1 through POD4. CONCLUSIONS: The data indicate that treatment with Cyclosporine A but not cycloheximide has a positive effect on cerebral recovery following HCA. Whether CsA results in inhibition of neuronal apoptosis, and/or inhibits release of cytokines and thereby reduces postischemic cerebral edema remains to be elucidated. The neuroprotective effect of CsA, if confirmed in further studies, would make its clinical application conceivable.

In situ detection of apoptosis in normal pressure glaucoma. a preliminary examination.

Retinal ganglion cell (RGC) neurons are believed to die via apoptosis in human primary and secondary open-angle glaucoma. Previous studies have relied solely on the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate [UTP]-biotin nick end-labeling) method of detecting DNA fragmentation to identify apoptotic nuclei. However, it is now clear that the TUNEL method cannot distinguish between the single- and double-strand DNA breaks that can be a feature of both apoptosis and necrosis. We have developed a double fluorescent labeling method that simultaneously combines in situ end-labeling (ISEL) to detect DNA fragmentation followed by staining with a cyanine dye, YOYO-1, to visualize apoptotic chromatin condensation. This allows for the unequivocal identification of an apoptotic nucleus. Our preliminary data obtained from one case of normal pressure glaucoma suggests that RGC neurons may die via apoptosis when intraocular pressure is not elevated.

Increased caspase 3 and Bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in Parkinson's disease.

In situ end labeling combined with YOYO staining was used to mark apoptotic DNA fragmentation and chromatin condensation respectively in human postmortem brain sections. Increased numbers of apoptotic neuronal nuclei were identified in the Parkinson's disease (PD) nigra compared with age-matched controls. Caspase 3 and Bax showed increased immunoreactivity in melanized neurons of the PD nigra compared with controls. Importantly, GAPDH nuclear accumulation was also observed in the PD nigra, suggesting apoptotic rather than necrotic cell death. Interestingly, both Lewy bodies and the intranuclear Marinesco's bodies were GAPDH immunoreactive in the PD brain.

Reduced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3-phosphate dehydrogenase to a dimer.

Antisense oligonucleotides against the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are able to reduce some forms of apoptosis. In those forms, overall GAPDH levels increase and the enzyme accumulates in the nucleus. The monoamine oxidase B (MAO-B) inhibitor, (-)-deprenyl (DEP), its metabolite (-)-desmethyldeprenyl, and a tricyclic DEP analog, CGP3466, can reduce apoptosis independently of MAO-B inhibition and have been found to bind to GAPDH. We used neuronally differentiated PC12 cells to show that DEP, DES, and CGP3466 reduce apoptosis caused by serum and nerve growth factor withdrawal over the concentration range of 10(-) to 10(-13) M. We provide evidence that the DEP-like compounds bind to GAPDH in the PC12 cells and that they prevent both the apoptotic increases in GAPDH levels and nuclear accumulation of GAPDH. In vitro, the compounds enhanced the conversion of NAD(+) to NADH by GAPDH in the presence of AUUUA-rich RNA and converted GAPDH from its usual tetrameric form to a dimeric form. Using cell lysates, we found a marked increase in rates of NAD(+) to NADH conversion in early apoptosis, which was returned toward control values by the DEP-like compounds. Accordingly, the DEP-like compounds appear to decrease glycolysis by preventing the GAPDH increases in early apoptosis. GAPDH dimer may not have the capacity to contribute to apoptosis in a similar manner to the tetramer, which might account for the antiapoptotic capacity of the compounds. These actions on GAPDH, rather than MAO-B inhibition, may contribute to the improvements in Parkinson's and Huntington's diseases found with DEP treatment.

Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic enzyme that plays a key role in energy metabolism. GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in the glycolytic pathway. As part of the conversion, GAPDH converts NAD+ to the high-energy electron carrier NADH. GAPDH has been referred to as a "housekeeping" protein and based on the view that GAPDH gene expression remains constant under changing cellular conditions, the levels of GAPDH mRNA have frequently been used to normalize northern blots. In recent years, that view has changed since GAPDH is now known to contribute to a number of diverse cellular functions unrelated to glycolysis. Normative functions of GAPDH now include nuclear RNA export, DNA replication, DNA repair, exocytotic membrane fusion, cytoskeletal organization and phosphotransferase activity. Pathologically, GAPDH has been implicated in apoptosis, neurodegenerative disease, prostate cancer and viral pathogenesis (see Sirover (1999) for a recent review of GAPDH functions). Most recently, it has been shown that GAPDH is a target for deprenyl related compounds (Carlile et al., 2000; Kragten et al., 1998) and may contribute to the neuroprotection offered by those compounds.

Confocal microscopy as a tool to examine DNA fragmentation, chromatin condensation and other apoptotic changes in Parkinson's disease.

There is considerable controversy regarding the possibility that nigral dopaminergic neurons may die via apoptosis in Parkinson's disease. It is now clear that both single- and/or double-stranded DNA breaks can be generated in the apoptotic degradative process. Since these breaks may also be present in necrotic cell death, in situ end labeling cannot be used in isolation to identify apoptotic neurons. We have developed a fluorescent double-labeling method that combines in situ end labeling with the simultaneous visualization of chromatin condensation. When viewed with laser confocal scanning microscopy, the structural detail of the nucleus is provided to unequivocally identify apoptotic nuclei.

Mitochondrial permeability in neuronal death: possible relevance to the pathogenesis of Parkinson's disease.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes catecholaminergic nerve cell loss and a syndrome similar to Parkinson's disease (PD). The metabolite of MPTP, MPP(+) (1-methyl-4-phenylpyridinium), decreases mitochondrial complex I activity similar to that in the PD nigra. Opening of a multi-protein, mitochondrial membrane pore constitutes a critical decisional event in some forms of apoptosis. We review recent findings showing that the permeability transition pore (PTP) opening caused by a decrease in the mitochondrial membrane potential (DeltaPsi(M)) contributes to MPP(+)-induced apoptosis. The reduction in DeltaPsi(M) appears to result from decreased proton pumping at complex I and therefore decreased complex I activity may also contribute to apoptosis in PD.

A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson's disease.

In situ end-labeling (ISEL) has become a widely used method to determine whether cells die via apoptosis by detecting double-stranded DNA breaks that are the result of endonuclease digestion. The enzyme terminal deoxynucleotidyl transferase can be used to label the digested 3'-OH ends of DNA with biotin-, digoxigenin-, or fluorescent probe-conjugated nucleotides. However, both single-stranded and double-stranded DNA breaks can be labeled by this method and therefore ISEL cannot unequivocally demonstrate apoptosis when used alone. We have developed a fluorescent double-labeling method using ISEL combined with the cyanine dye YOYO-1 that binds to DNA. When combined with confocal laser microscopy and deconvolution analysis, YOYO-1 can demonstrate the presence or absence of nuclear chromatin condensation and thus confirm that ISEL-positive nuclei are indeed apoptotic. Preliminary findings indicate that dopaminergic neurons in the substantia nigra compacta die via apoptosis in Parkinson's disease.

In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining.

The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to generate a dose-dependent cell death of dopaminergic nigral neurons in the C57B1 mouse. Mice were injected with a total cumulative dose of 150 mg/kg of MPTP delivered over five days and killed at different time points both during and after the toxin injections. Two independent histological methods were used to determine whether the dopaminergic nigral neurons died via an apoptotic mechanism. In situ end-labelling with terminal deoxynucleotidyl transferase was used on paraformaldehyde-fixed, serial, frozen sections to identity cells with double-stranded DNA breaks. Apoptotic cell death was found to be initiated within 72 h of the first injection of the neurotoxin and peaked 24 h after the final MPTP injection. The metachromatic fluorochrome, Acridine Orange, was used on alternate sections to provide structural confirmation of the nuclear chromatin "clumping" considered to be representative of apoptosis. Confocal laser imaging combined with deconvolution techniques was used to resolve the fluorescent signal emitted by the in situ Acridine Orange-DNA complexes. The number of Acridine Orange-stained nuclei demonstrating chromatin clumping was identical to that of the positive in situ end-labelled nuclei observed over a 25 day period. Based upon these two independent methods of assessing apoptosis in situ, we conclude that a 150 mg/kg dose of MPTP can elicit apoptotic cell death in nigral dopaminergic neurons of the C57B1 mouse.

Apoptosis in neurodegenerative disorders: potential for therapy by modifying gene transcription.

Apoptotic, rather than necrotic, nerve cell death now appears as likely to underlie a number of common neurological conditions including stroke, Alzheimer's disease, Parkinson's disease, hereditary retinal dystrophies and Amyotrophic Lateral Sclerosis. Apoptotic neuronal death is a delayed, multistep process and therefore offers a therapeutic opportunity if one or more of these steps can be interrupted or reversed. Research is beginning to show how specific macromolecules play a role in determining the apoptotic death process. We are particularly interested in the critical nature of gradual mitochondrial failure in the apoptotic process and propose that a maintenance of mitochondrial function through the pharmacological modulation of gene expression offers an opportunity for the effective treatment of some types of neurological dysfunction. Our research into the development of small diffusible molecules that reduce apoptosis has grown from studies of the irreversible MAO-B inhibitor (-)-deprenyl. (-)-Deprenyl can reduce neuronal death independently of MAO-B inhibition even after neurons have sustained seemingly lethal damage. (-)-Deprenyl can also influence the process outgrowth of some glial and neuronal populations and can reduce the concentrations of oxidative radicals in damaged cells at concentrations too small to inhibit MAO. In accord with earlier work of others, we showed that (-)-deprenyl alters the expression of a number of mRNAs or of proteins in nerve and glial cells and that the alterations in gene expression/protein synthesis are the result of a selective action on transcription. The alterations in gene expression/protein synthesis are accompanied by a decrease in DNA fragmentation characteristic of apoptosis and the death of responsive cells. The onco-proteins Bcl-2 and Bax and the scavenger proteins Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD-2) are among the 40-50 proteins whose synthesis is altered by (-)-deprenyl. Since mitochondrial membrane potential correlates with mitochondrial ATP production, we have used confocal laser imaging techniques in living cells to show that the transcriptional changes induced by (-)-deprenyl result in a maintenance of mitochondrial membrane potential, a decrease in intramitochondrial calcium and a decrease in cytoplasmic oxidative radical levels. We therefore propose that (-)-deprenyl acts on gene expression to maintain mitochondrial function and decrease cytoplasmic oxidative radical levels and thereby reduces apoptosis. An understanding of the molecular steps by which (-)-deprenyl selectively alters transcription may lead to the development of new therapies for neurodegenerative diseases.

(-)-Deprenyl reduces neuronal apoptosis and facilitates neuronal outgrowth by altering protein synthesis without inhibiting monoamine oxidase.

(-)-Deprenyl stereospecifically reduces neuronal death even after neurons have sustained seemingly lethal damage at concentrations too small to cause monoamine oxidase-B (MAO-B) inhibition. (-)-Deprenyl can also influence the process growth of some glial and neuronal populations and can reduce the concentrations of oxidative radicals in damaged cells at concentrations too small to inhibit MAO. In accord with the earlier work of others, we showed that (-)-deprenyl alters the expression of a number mRNAs or proteins in nerve and glial cells and that the alterations in gene expression/protein synthesis are the result of a selective action on transcription. The alterations in gene expression/protein synthesis are accompanied by a decrease in DNA fragmentation characteristic of apoptosis and the death of responsive cells. The onco-proteins Bcl-2 and Bax and the scavenger proteins Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) are among the 40-50 proteins whose synthesis is altered by (-)-deprenyl. Since mitochondrial ATP production depends on mitochondrial membrane potential (MMP) and mitochondrial failure has been shown to be one of the earliest events in apoptosis, we used confocal laser imaging techniques in living cells to show that the transcriptional changes induced by (-)-deprenyl are accompanied by a maintenance of mitochondrial membrane potential, a decrease in intramitochondrial calcium and a decrease in cytoplasmic oxidative radical levels. We therefore propose that (-)-deprenyl acts on gene expression to maintain mitochondrial function and to decrease cytoplasmic oxidative radical levels and thereby to reduce apoptosis. An understanding of the molecular steps by which (-)-deprenyl selectively alters transcription may contribute to the development of new therapies for neurodegenerative diseases.

Neurons express ciliary neurotrophic factor mRNA in the early postnatal and adult rat brain.

The regional and subcellular localization in the central nervous system (CNS) of postnatal day 5, day 15, and adult rats of ciliary neurotrophic factor (CNTF) mRNA was examined by in situ hybridization with biotinylated riboprobes. Probe specificity was determined by Northern blot analysis of poly(A)+ RNA extracted from adult rat brain using digoxigenin labeled riboprobes and chemiluminescent detection. Both a 4 kb and a 1.2 kb transcript were detected in the cortex and brainstem. In situ hybridization revealed that CNTF mRNA was widely distributed in neurons and glia throughout the CNS at each of the developmental time points. The density of the neuronal hybridization signal was found to be greater in neuronal nuclei than in their cytoplasm. In the nucleus of most neurons, CNTF mRNA distribution was concentrated in a perinucleolar fashion. Alternate sections from the same animals, which were incubated with a specific polyclonal antibody against a CNTF peptide fragment, revealed that both neurons and glia in postnatal day 5, day 15, and adult rat brain were immunoreactive for CNTF.