Nadine A. Tatton

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 Parkinsons 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 Marinescos bodies were GAPDH immunoreactive in the PD brain.

Apoptosis in Parkinson's disease: signals for neuronal degradation.

Controversy has surrounded a role for apoptosis in the loss of neurons in Parkinsons 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

Spatial distribution of Bax and Bcl-2 in osteocytes after bone fatigue: complementary roles in bone remodeling regulation?

Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)‐mediated deoxyuridine triphosphate (dUTP)‐biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl‐2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl‐2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl‐2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl‐2 expression, the antiapoptotic signal, reached its greatest level at some distance (1‐2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively “walled in” by a surrounding halo of surviving osteocytes actively expressing Bcl‐2. Thus, the expression pattern of apoptosis‐inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue.

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

Summary. Deprenyl and other propargylamines are clinically beneficial in Parkinsons 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.

Hypothesis for a common basis for neuroprotection in glaucoma and Alzheimer's disease: anti-apoptosis by alpha-2-adrenergic receptor activation.

Recent studies have suggested glaucomatous loss of retinal ganglion cells and their axons in Alzheimers disease. Amyloid beta peptides and phosphorylated tau protein have been implicated in the selective regional neuronal loss and protein accumulations characteristic of Alzheimers disease. Similar protein accumulations are not present on glaucomatous retinal ganglion cells. Neurons die in both Alzheimers disease and glaucoma by apoptosis, although the signaling pathways for neuronal degradation appear to differ in the two diseases. Alzheimers disease features a loss of locus ceruleus noradrenergic neurons, which send axon terminals to the brain regions suffering neuronal apoptosis and results in reductions in noradrenaline in those regions. Activation of alpha-2 adrenergic receptors reduces neuronal apoptosis, in part through a protein kinase B (Akt)-dependent signaling pathway. Loss of noradrenaline innervation facilitates neuronal apoptosis in Alzheimers disease models and may act similarly in glaucoma. Alpha-2 adrenergic receptor agonists offer the potential to slow the neuronal loss in both diseases by compensating for lost noradrenaline innervation.

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.

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.

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.

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 Parkinsons 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.