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Controversy has surrounded a role for apoptosis in the loss of neurons in Parkinson's disease (PD). Although a variety of evidence has supported an apoptotic contribution to PD neuronal loss particularly in the nigra, two factors have weighed against general acceptance: (1) limitations in the use of in situ 3′ end labeling techniques to demonstrate nuclear DNA cleavage; and (2) the insistence that a specific set of nuclear morphological features be present before apoptotic death could be declared. We first review the molecular events that underlie apoptotic nuclear degradation and the literature regarding the unreliability of 3′ DNA end labeling as a marker of apoptotic nuclear degradation. Recent findings regarding the multiple caspase‐dependent or caspase‐independent signaling pathways that mediate apoptotic nuclear degradation and determine the morphological features of apoptotic nuclear degradation are presented. The evidence shows that a single nuclear morphology is not sufficient to identify apoptosis and that a cytochrome c, pro–caspase 9, and caspase 3 pathways is operative in PD nigral apoptosis. BAX‐dependent increases in mitochondrial membrane permeability are responsible for the release of mitochondrial factors that signal for apoptotic degradation, and increased BAX levels have been found in a subset of PD nigral neurons. Studies using immunocytochemistry in PD postmortem nigra have begun to define the premitochondrial apoptosis signaling pathways in the disease. Two, possibly interdependent, pathways have been uncovered: (1) a p53–glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)–BAX pathway; and (2) FAS receptor–FADD–caspase 8–BAX pathway. Based on the above, it seems unlikely that apoptosis does not contribute to PD neuronal loss, and the definition of the premitochondrial signaling pathways may allow for the development and testing of an apoptosis‐based PD therapy. Ann Neurol 2003;53 (suppl 3):S61–S72

Controversy has surrounded a role for apoptosis in the loss of neurons in Parkinson's disease (PD). Although a variety of evidence has supported an apoptotic contribution to PD neuronal loss particularly in the nigra, two factors have weighed against general acceptance: (1) limitations in the use of in situ 3' end labeling techniques to demonstrate nuclear DNA cleavage; and (2) the insistence that a specific set of nuclear morphological features be present before apoptotic death could be declared. We first review the molecular events that underlie apoptotic nuclear degradation and the literature regarding the unreliability of 3' DNA end labeling as a marker of apoptotic nuclear degradation. Recent findings regarding the multiple caspase-dependent or caspase-independent signaling pathways that mediate apoptotic nuclear degradation and determine the morphological features of apoptotic nuclear degradation are presented. The evidence shows that a single nuclear morphology is not sufficient to identify apoptosis and that a cytochrome c, pro–caspase 9, and caspase 3 pathways is operative in PD nigral apoptosis. BAX-dependent increases in mitochondrial membrane permeability are responsible for the release of mitochondrial factors that signal for apoptotic degradation, and increased BAX levels have been found in a subset of PD nigral neurons. Studies using immunocytochemistry in PD postmortem nigra have begun to define the premitochondrial apoptosis signaling pathways in the disease. Two, possibly interdependent, pathways have been uncovered: (1) a p53–glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–BAX pathway; and (2) FAS receptor–FADD–caspase 8–BAX pathway. Based on the above, it seems unlikely that apoptosis does not contribute to PD neuronal loss, and the definition of the premitochondrial signaling pathways may allow for the development and testing of an apoptosis-based PD therapy. Ann Neurol 2003;53 (suppl 3):S61–S72

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 10−13M. 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.

MPP+inhibits mitochondrial complex I and α-ketoglutarate dehydrogenase causing necrosis or apoptosis of catecholaminergic neurons. Low glucose levels or glycolytic blockade has been shown to potentiate MPP+toxicity. We found that MPP+caused concentration-dependent apoptosis of neuronally differentiated PC12 cells and that glucose, but not pyruvate, supplementation reduced apoptosis. Oligomycin concentrations sufficient to inhibit ATP synthase blocked the decreased apoptosis afforded by glucose supplementation. Laser-scanning confocal microscope imaging of chloromethyl-tetramethylrosamine methyl ester fluorescence to estimate ΔΨMshowed that MPP+and atractyloside reduced ΔΨM, while cyclosporin A (CSA) and glucose supplementation reversed decreases in ΔΨMcaused by MPP+. Oligomycin blocked the effect of glucose supplementation on ΔΨM. These findings show that (i) MPP+-induced and atractyloside-induced apoptosis are associated with reduced ΔΨM; (ii) CSA maintains ΔΨMand reduces MPP+-induced apoptosis; and (iii) glucose supplementation maintains ΔΨM,likely by glycolytic ATP-dependent proton pumping at ATP synthase and reduces MPP+-induced apoptosis.

There is accumulating evidence that mitochondrial membrane potential (ΔΨM) is reduced in aged cells. In addition, a decrease of ΔΨMhas been shown to be an early event in many forms of apoptosis. Here we use a mitochondrial potentiometric dye with in situlaser scanning confocal microscopic (LSCM) imaging to demonstrate that ΔΨMis dramatically decreased in both the p53-overexpressing, senescent EJ tumor cells and in pre-apoptotic PC12 cells compared to controls. Treatment with cyclosporin A (CSA), which facilitates closure of the mitochondrial permeability transition pore (PTP), was able to reverse the decrease in ΔΨMin pre-apoptotic PC12 cells but not in the senescent EJ-p53 cells. The capacity to prevent dissipation of ΔΨMin response to agents that facilitate PTP closure may differentiate cells entering apoptosis from those participating in senescence. Therefore, regulation of the closure of the mitochondrial PTP in the presence of decreased ΔΨMmay be a decisional checkpoint in distinguishing between growth arrest pathways.

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.

Apoptotic cell death has been shown to constitute the terminal process in some neurodegenerative diseases, notably Alzheimer's disease and Parkinson's disease (PD). A decrease in mitochondrial membrane potential (ΔψM) causing opening of the permeability transition pore (PTP) in mitochondrial membranes has been implicated as a critical effector of apoptosis in a variety of non‐neural cells. Opening of the PTP leads to the release of so‐called apoptosis initiation factors that induce the degradative events of apoptosis, such as nuclear chromatin condensation and DNA fragmentation. We have extended those findings to a neuronal model of apoptosis caused by trophic withdrawal, by showing that a decrease in ΔψMis an early event occurring 2 to 6 hours before the degradative events of apoptosis. A deficiency in mitochondrial complex I activity has been demonstrated in the substantia nigra of postmortem brains and several peripheral tissues obtained from PD patients. Because ΔψMis generated by the pumping of protons out across the inner mitochondrial membrane at the mitochondrial complexes, particularly complex I, we hypothesized that the decrease in complex activity could result in a decrease in ΔψMthat would render PD substantia nigra neurons vulnerable to apoptosis. In preliminary studies, we have found a decrease in ΔψMin fibroblasts obtained from some PD patients. If a decrease in ΔψMconsequent on decreased complex activity is an intrinsic defect in some PD patients, it would open a number of new avenues for the reduction of neuronal apoptosis in PD. The oncoprotein BCL‐2 and the scavenger protein SOD‐1 have been shown to reduce apoptosis by facilitating closure of the PTP. A number of agents have been shown to maintain BCL‐2 and/or SOD‐1 synthesis in damaged nerve cells and thereby reduce apoptosis. Other agents, such as cyclosporin A and some benzodiazepine receptor‐binding agents, have been found to act directly on the PTP to reduce apoptosis. Accordingly, agents that maintain ΔψMand PTP closure may offer new and effective means of treating neurodegenerative apoptosis.

METABOLIC hypofunction is a common finding in a number of neurodegenerative diseases, including Alzheimer's disease (AD). The strong linkage between the amyloid precursor protein (APP) and AD led us to examine whether over-expression of this protein in CNS-type cells had an effect on mitochondria. We found abnormal morphology in mitochondria of the neuroectodermal progeny of P19 cells stably transfected with human APP751. In addition, the mitochondria of APP-transfected clones had a decreased mitochondrial membrane potential. These changes were independent of Aβ toxicity and distinct from complex I inhibition. Our results have important implications for the earliest events in the pathophysiology of AD and, by extrapolation, for intervention therapies.

(-)-Deprenyl previously was shown to increase the survival of rat facial motoneurons (FMns) after a loss of muscle-derived trophic support caused by axotomy at Postnatal Day 14 (P14) and to increase reactive astrogliosis after traumatic damage to the adult rat striatum. We estimated reactive astrogliosis in facial nuclei at 1, 3, 7, 14, and 21 days after transection of the facial nerve at P14 by two methods: first, by measuring the relative optical density (OD) of GFAP immunoreaction (GFAP-OD) in the facial nuclei and second by determining the relative area of GFAP immunoreactivity (GFAP-AREA) in the same nuclei. Both measures were taken for multiple immunoreacted sections through the length of each facial nuclei by using a control half section at the same brain stem level taken from an unlesioned, age-matched animal. The experimental and control facial nuclear half sections were coimmunoreacted using the "glued" half brain stem method. The facial nerve transections served to axotomize all of the FMns in the ipsilateral facial nuclei. The numbers of surviving FMns were examined at the same time points as above using counts of Nissl-stained somata from serial sections taken through each facial nucleus. We found that FMn loss occurred rapidly after axotomy in saline-treated animals and could be best fitted with a decaying exponential relationship (time constant 2.7 days). In the saline-treated animals, the FMn loss plateaued between 7 and 14 days at 74.8%, and 47% of the FMns were found to be lost within 3 days. Increases in the facial nuclear GFAP-OD values and GFAP-AREA values were evident as early as 1 day following axotomy (2.5 and 3.3 times normal, respectively) and reached maximal levels by 7 days (5.7 and 37.6 times normal, respectively). The administration of (-)-deprenyl slowed the loss of the FMns by 24-48 h (time constant 3.9 days) and increased the number of surviving FMns at 21 days by 2.1 times. Treatment with (-)-deprenyl was found to significantly increase GFAP-OD and GFAP-AREA at Day 1 by 71 and 32%, respectively, and at Day 3 by 22 and 27%, respectively. In contrast, it decreased GFAP-OD and GFAP-AREA by 42 and 19%, respectively, at Day 7, and by 20 and 12%, respectively at Day 21. Accordingly, as estimated by both measures, the drug increases reactive astrogliosis in the facial nucleus during the first 3 days after facial nerve transection and decreases the gliosis thereafter. The increased astrogliosis at Days 1 and 3 may contribute to the increased survival of the axotomized, immature FMns caused by (-)-deprenyl over the same time period.

The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH–PML interaction may be involved in the regulation of apoptosis.