Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease.

Although the cause of Parkinson's disease (PD) is unknown, data suggest roles for environmental factors that may sensitize dopaminergic neurons to age-related dysfunction and death. Based upon epidemiological data suggesting roles for dietary factors in PD and other age-related neurodegenerative disorders, we tested the hypothesis that dietary folate can modify vulnerability of dopaminergic neurons to dysfunction and death in a mouse model of PD. We report that dietary folate deficiency sensitizes mice to MPTP-induced PD-like pathology and motor dysfunction. Mice on a folate-deficient diet exhibit elevated levels of plasma homocysteine. When infused directly into either the substantia nigra or striatum, homocysteine exacerbates MPTP-induced dopamine depletion, neuronal degeneration and motor dysfunction. Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in human dopaminergic cells exposed to the pesticide rotenone or the pro-oxidant Fe(2+). The adverse effects of homocysteine on dopaminergic cells is ameliorated by administration of the antioxidant uric acid and by an inhibitor of poly (ADP-ribose) polymerase. The ability of folate deficiency and elevated homocysteine levels to sensitize dopaminergic neurons to environmental toxins suggests a mechanism whereby dietary folate may influence risk for PD.     

Cell-death mechanisms in the IPLB-LdFB insect cell line: a nuclear located Bcl-2-like molecule as a possible controller of 2-deoxy-<Emphasis Type="SmallCaps">D</Emphasis>-ribose-mediated DNA fragmentation

In the IPLB-LdFB insect cell line, oncosis and apoptosis are the two pre-mortal processes, whereas necrosis is the post-mortem condition. As found in mammals, adenosine triphosphate depletion of insect cells by oligomycin A induces oncosis. The apoptotic inducer 2-deoxy-D-ribose (dRib) provokes cell death through an intrinsic apoptotic pathway similar to that observed in mammalian models and results in oligonucleosomal DNA fragmentation. The addition to insect cells of an anti-Bcl-2 polyclonal antibody known to prevent dRib-mediated apoptosis abolishes DNA fragmentation, whereas cytochrome c release and the increase in a caspase 3-like activity are still detectable. These and previous findings suggest a double role for the Bcl-2-like molecule in IPLB-LdFB, i.e. the maintenance of mitochondrial integrity and the control of apoptotic machinery at the nuclear level. This work was supported by a MIUR (Italy) grant to E.O.     

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.     

PDGF and TGF-beta partially prevent 2-deoxy-D-ribose-induced apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar

The IPLB-LdFB cell line from the fat body of the insect Lymantria dispar shows the presence of immunoreactive, platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 molecules, as well as the corresponding plasma membrane-like receptors, i.e. PDGFR-alpha, PDGFR-beta and TGFR-beta type II. Cytofluorimetric and morphological studies reveal that the reducing sugar 2-deoxy-D-ribose (dRib), an apoptotic agent for human cells, induces apoptosis in a concentration- and time-dependent manner even in IPLB-LdFB cells. PDGF-AB and TGF-beta1 partially counteract the effect of dRib, indicating a survival role of these factors in this apoptotic model of insect cells.     

Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells

Green tea, owing to its beneficial effect on health, is becoming more and more popular worldwide. (-)-Epigallocatechin-3-gallate (EGCG), the main ingredient of green tea polyphenols, is a known protective effect on injured neurons in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. Paraquat (PQ) is a widely used herbicide that possesses a similar structure to MPP(+) and is toxic to mesencephalic dopaminergic neurons. In the present study, PQ-injured PC12 cells were chosen as an in vitro cell model of Parkinson's disease and the neuroprotective effects of EGCG were investigated. The results showed that EGCG attenuated apoptosis of PC12 cells induced by PQ. The possible mechanism may be associated with maintaining mitochondrial membrane potential, inhibiting caspase-3 activity and downregulating the expression of pro-apoptotic protein Smac in cytosol. The present study supports the notion that EGCG could be used as a neuroprotective agent for treatment of neurodegenerative diseases.     

Survivin: a target from brain cancer to neurodegenerative disease

Apoptosis is an important contributing factor during neuronal death in a variety of neurodegenerative disorders, including multiple sclerosis, Parkinson's disease and sciatic nerve injury. Whereas several clinical and preclinical studies have focused on the neuroprotective effects of caspase inhibitors, their clinical benefits are still unclear. Here, we discuss novel alternative strategies to protect neuronal cells from apoptotic death using members of the inhibitors of apoptosis (IAP) family. We specifically review the different roles of survivin, which is an important member of the IAP family that serves a dual role in the inhibition of apoptosis as well as a vital role in mitosis and cell division. Due to the various roles of survivin during cell division and apoptosis, targeting this protein illustrates a new therapeutic window for the treatment of neurodegenerative diseases.     

New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by an F.A.R. grant from the University of Modena and Reggio Emilia (D.M. and E.O.) and by an “Experimental approaches to the study of evolution” grant from the Department of Animal Biology of the University of Modena and Reggio Emilia (D.M.).     

Involvement of PI 3-kinase, PKA and PKC in PDGF- and TGF-beta-mediated prevention of 2-deoxy-D-ribose-induced apoptosis in the insect cell line, IPLB-LdFB

Activation of phosphatidylinositol (PI) 3-kinase, protein kinase A (PKA) and protein kinase C (PKC) is associated with the survival effect elicited by PDGF-AB and TGF-beta1 against the apoptotic inducer 2-deoxy-D-ribose (dRib) in the fat body cell line, IPLB-LdFB, from the insect Lymantria dispar. dRib induces apoptosis and provokes mitochondrial membrane depolarization (MMD). The antioxidant N -acetyl-L-cysteine annuls only the first effect. These findings suggest that apoptosis and MMD are provoked by two different mechanisms, and that dRib induces apoptosis by oxidative stress.     

Neuroprotective effects of SCM198 on 6-hydroxydopamine-induced behavioral deficit in rats and cytotoxicity in neuronal SH-SY5Y cells

6-Hydroxydopamine (6-OHDA), one of the most investigated Parkinson's disease neurotoxins, is widely used to study mechanisms of cell death in dopaminergic neurons. In the present study, we demonstrated that SCM198, a new compound based on the active component of Herba leonuri, significantly reduced 6-OHDA-induced cell death in dopaminergic SH-SY5Y cells and attenuated apomorphine-elicited rotational behavior in 6-OHDA-lesioned rats. Pretreatment with SCM198 (0.001, 0.01, 0.1, 1, and 10 μM) concentration-dependently increased the cell viability as measured in MTT and LDH leakage assays compared with 6-OHDA-injured cells. Tocopherol, an antioxidant used as positive control, had similar effect at 10 μM to SCM198 1 μM. Furthermore, we assessed oxidative stress and subsequent apoptosis, the critical players in dopaminergic neurodegeneration, with 0.1, 1, and 10 μM of SCM198 in SH-SY5Y cells exposed to 6-OHDA. Pretreatment with SCM198 significantly increased antioxidant enzyme superoxide dismutase activity, ameliorated intracellular reactive oxygen species generation, prevented the dissipation of mitochondrial membrane potential, decreased apoptotic cell death in Hoechst 33258 staining, as well as down-regulated Bax and up-regulated Bcl-2 in both mRNA and protein levels compared with 6-OHDA damaged cells. Moreover, intragastrical administration of SCM198 (18 or 60 mg kg?1 day?1) for 4 weeks significantly ameliorates apomorphine-induced contralateral rotations in 6-OHDA-lesioned rats. These results support the neuroprotective effects of SCM198 against 6-OHDA-induced toxicity in vivo and in vitro with the underlying mechanisms of inhibiting oxidative stress and apoptosis. Therefore we suggest that SCM198 might provide a useful therapeutic strategy for neurodegenerative diseases such as Parkinson's disease.     

Cell death mechanisms in neurodegeneration

Progressive cell loss in specific neuronal populations often associated with typical cytoskeletal protein aggregations is a pathological hallmark of neurodegenerative disorders, but the nature, time course and molecular causes of cell death and their relation to cytoskeletal pathologies are still unresolved. Apoptosis or alternative pathways of cell death have been discussed in Alzheimer's disease and other neurodegenerative disorders. Apoptotic DNA fragmentation in human brain as a sign of neuronal injury is found too frequent as to account for continous neuron loss in these slowly progressive processes. Morphological studies revealed extremely rare apoptotic neuronal death in Alzheimer's disease but yielded mixed results for Parkinson's disease and other neurodegenerative disorders. Based on recent data in human brain, as well as in animal and cell culture models, a picture is beginning to emerge suggesting that, in addition to apoptosis, other forms of programmed cell death may participate in neurodegeneration. Better understanding of the molecular players will further elucidate the mechanisms of cell death in these disorders and their relations to cytoskeletal abnormalities. Susceptible cell populations in a proapoptotic environment show increased vulnerability towards multiple noxious factors discussed in the pathogenesis of neurodegeneration. In conclusion, although many in vivo and in vitro data are in favor of apoptosis involvement in neurodegenerative processes, there is considerable evidence that very complex events may contribute to neuronal death with possible repair mechanisms, the elucidation of which may prove useful for future prevention and therapy of neurodegenerative disorders.     

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.     

Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.     

Prevention of paraquat-induced apoptosis in human neuronal SH-SY5Y cells by lipocalin-type prostaglandin D synthase

Paraquat is a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant 1-methyl-4-phenyl-pyridine and acts as a potential etiologic factor for the development of Parkinson's disease. In this study, we investigated the protective roles of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) against paraquat-mediated apoptosis of human neuronal SH-SY5Y cells. The treatment of SH-SY5Y cells with paraquat decreased the intracellular GSH level, and enhanced the cell death with elevation of the caspase activities. L-PGDS was expressed in SH-SY5Y cells, and its expression was enhanced with the peak at 2?h after the initiation of the treatment with paraquat. Inhibition of PGD? synthesis and exogenously added PGs showed no effects regarding the paraquat-mediated apoptosis. SiRNA-mediated suppression of L-PGDS expression in the paraquat-treated cells increased the cell death and caspase activities. Moreover, over-expression of L-PGDS suppressed the cell death and caspase activities in the paraquat-treated cells. The results of a promoter-luciferase assay demonstrated that paraquat-mediated elevation of L-PGDS gene expression occurred through the NF-κB element in the proximal promoter region of the L-PGDS gene in SH-SY5Y cells. These results indicate that L-PGDS protected against the apoptosis in the paraquat-treated SH-SY5Y cells through the up-regulation of L-PGDS expression via the NF-κB element. Thus, L-PGDS might potentially serve as an agent for prevention of human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.     

Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide

We examined the hypothesis that sodium nitroprusside (SNP) produces cell death in cardiomyocytes through generation of H(2)O(2). Embryonic chick cardiomyocytes in culture were treated with SNP, and cell viability was assessed by trypan blue, MTT assay, and fluorescent activated cell sorting (FACS) analysis. SNP for 24 h induced a significant (P < 0.001) dose-dependent loss of cell viability. On MTT assay, the half-maximal effective concentration was 0.53 mM (confidence interval 0.45-0.59 mM). SNP-treated cardiomyocytes displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated SNP-induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by propidium iodide (PI) staining of permeabilized cells, increased significantly (P < 0.05) after 0.5 mM SNP for 24 h. The proportion of apoptotic cells, characterized by dual staining of intact cardiomyocytes with fluorescein diacetate and PI, was significantly (P < 0.05) increased after treatment with 0.5 mM SNP for 24 h. SNP metabolism and NO production was suggested by the significant (P < 0.05) increase in nitrite generation in the media with 0.5 mM SNP compared with control. SNP-mediated H(2)O(2) production was implicated in the mechanism of SNP-induced cell death. First, SNP produced a significant (P < 0.05) increase in H(2)O(2) detected in the media after 6 or 24 h of SNP treatment. Second, catalase completely blocked the reduction of cell viability induced by 0.1 mM SNP and significantly (P < 0.05) blunted the effect of 0.5 mM SNP. In contrast, the iron chelator deferoxamine did not alter SNP-induced loss of cell viability. FACS analysis showed that the combination of low concentrations of H(2)O(2) (10(-8) M) that did not alter cell viability augmented SNP-induced apoptosis. In contrast, the amount of necrotic cell death was unchanged by the combination of H(2)O(2) and SNP. H(2)O(2) plus SNP produced a dramatic alteration in cell structure with greater membrane bleb formation, shrunken cells, and more intense cytosolic acridine orange staining and nuclear fragmentation than either agent alone. These data indicate the vulnerability of cardiomyocytes to SNP and suggest the involvement of H(2)O(2) in the pathogenesis of SNP-induced cardiomyocyte cell death. Establishing apoptosis as a component of the type of cell death induced by SNP permitted the recognition that SNP-induced apoptosis was increased by chronic treatment with low (subtoxic) concentrations of H(2)O(2).     

Type A monoamine oxidase is the target of an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, leading to apoptosis in SH-SY5Y cells

Mitochondrial monoamine oxidase (MAO) has been considered to be involved in neuronal degeneration either by increased oxidative stress or protection with the inhibitors of type B MAO (MAO-B). In this paper, the role of type A MAO (MAO-A) in apoptosis was studied using human neuroblastoma SH-SY5Y cells, where only MAO-A is expressed. An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, an MAO-A inhibitor, reduced membrane potential, DeltaPsim, in isolated mitochondria, and induced apoptosis in the cells, which 5-hydroxytryptamine, an MAO-A substrate, prevented. In contrast, beta-phenylethylamine, an MAO-B substrate, did not suppress the DeltaPsim decline by N-methyl(R)salsolinol. The binding of N-methyl(R)salsolinol to mitochondria was inhibited by clorgyline, a MOA-A inhibitor, but not by (-)deprenyl, an MAO-B inhibitor. RNA interference targeting MAO-A significantly reduced the binding of N-methyl(R)salsolinol with simultaneous reduction in the MAO activity. To examine the intervention of MAO-B in the apoptotic process, human MAO-B was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even although the activity and protein of MAO increased markedly. These results demonstrate a novel function of MAO-A in the binding of neurotoxins and the induction of apoptosis, which may account for neuronal cell death in neurodegenerative disorders, including Parkinson's disease.