Nigral and Striatal Comparative Study of the Neurotoxic Action of 1-Methyl-4-Phenylpyridinium Ion: Involvement of Dopamine Uptake System

Abstract: Microdialysis was used in a comparative study of the neurotoxic action of MPP⁺ in the absence or presence of nomifensine (20 µM) in the striatum and substantia nigra. Three different concentrations of MPP⁺ (1, 2.5, and 5 mM) were perfused for 15 min at 24 (day 1) and 48 h (day 2) after surgery. The dopamine basal value in the striatum was ～17 fmol/min. Nomifensine (20 µM) stimulated dopamine release to ～170 fmol/min. The increase of dopamine extracellular output in the striatum after MPP⁺ perfusion on day 1 was independent of the concentration of MPP⁺ perfused and of the absence or presence of nomifensine (20 µM), being ～2,500 fmol/min. The dopamine basal value in the substantia nigra was below the detection limit of our HPLC equipment. Nomifensine (20 µM) stimulated dopamine release to ～6.3 fmol/min. The increase of dopamine extracellular output in the substantia nigra was MPP⁺ dose-dependent (1 mM, 75 fmol/min; 2.5 mM, 150 fmol/min; and 5 mM, 250 fmol/min) and independent of the presence or absence of nomifensine. On day 2, the presence of nomifensine on day 1 produced a total protection against MPP⁺ (1 mM) perfusion in the striatum, which was not observed against MPP⁺ (5 mM). MPP⁺ (1 mM) did not produce any neurotoxic action in the substantia in the absence or presence of nomifensine. The MPP⁺ (2.5 mM) effect on dopamine extracellular output in the absence of nomifensine (20 µM) in the substantia nigra on day 2 was similar to that of MPP⁺ (1 mM) in the striatum. The presence of nomifensine (20 µM) partially prevented the neurotoxic effect of MPP⁺ (2.5 mM) on dopaminergic cell bodies/dendrites in the substantia nigra. The MPP⁺ (5 mM) effect on dopamine extracellular output was similar in both structures studied in the absence or presence of nomifensine on day 2. These results suggest that terminals in the striatum are more sensitive to the neurotoxicity of MPP⁺ than cell bodies/dendrites in the substantia nigra.     

Pituitary Adenylyl Cyclase-Activating Polypeptide and Nerve Growth Factor Use the Proteasome to Rescue Nerve Growth Factor-Deprived Sympathetic Neurons Cultured From Chick Embryos

Abstract: Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24–48 h. Uptake of [³H]norepinephrine ([³H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1–100 nM) restored [³H]NE uptake to 92 ± 8% of that of NGF-supported controls. Depolarization-induced [³H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3′,5′-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 ± 40 versus 38 ± 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 ± 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

Studies on the Characterization of the Inhibitory Mechanism of 4'-Alkylated 1-Methyl-4-Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles

Abstract: 1-Methyl-4-phenylpyridinium (MPP⁺), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′-alkylated analogues of MPP⁺. These derivatives had IC₅₀ values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC₅₀ values of corresponding 4′-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ～600-fold, versus only a 36-fold, increase in the IC₅₀ of MPP⁺ versus 4′-pentyl-MPP⁺, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′-decyl-MPP⁺ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol. In addition, the lipophilic anion TPB^? was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.     

Both nerve growth factor and high K concentrations support the survival of chick embryo sympathetic neurons : Evidence for a common mechanism of action

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K⁺ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K⁺, and survival was not increased when both NGF and K⁺ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K⁺ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K⁺ and NGF for the survival of cultured embryonic neurons.     

Sympathetic neuronal survival induced by retinal trophic factors

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti‐NGF (1 μg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1–50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin‐receptors TrkA, TrkB, or TrkC had no effect on RCM‐induced sympathetic neuron survival. The survival effects were not blocked by anti‐GDNF, anti‐TGFβ, and anti‐CNTF and were not mimicked by FGFb (0.1–10 nM). LY294002 at 50 μM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high‐molecular weight retinal secreted factor that supports sympathetic neurons in culture. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 13–23, 2002     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Selective degeneration of dopaminergic neurons by MPP+ and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. We used Drosophila primary neuronal culture to study degenerative effects of a well‐known PD toxin MPP⁺. Dopaminergic (DA) neurons were selectively degenerated by MPP⁺, whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post‐mitotic degeneration, not by inhibition of DA neuronal differentiation. We also found that MPP⁺‐mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP⁺‐toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan‐neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP⁺ toxicity was not rescued by D2 agonists. Our study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons

Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (～2 × 10^?11M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.     

Ionic behaviors and neuronal survival in developing ganglia. III. Studies with embryonic chick sympathetic neurons

We have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na⁺,K⁺-pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na⁺ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13–E18), NGF becomes less and less required for that control as the neurons gain a self-sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (～2 × 10^?11M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.     

DLP1-dependent mitochondrial fragmentation mediates 1-methyl-4-phenylpyridinium toxicity in neurons: implications for Parkinson's disease

Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease (PD) can be modeled by the administration of the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP⁺). Because abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP⁺ on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP⁺ in neuronal cells. In SH‐SY5Y cells, MPP⁺ causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP⁺‐induced mitochondrial fragmentation. Notably, this approach partially rescues MPP⁺‐induced decline in ATP levels and ATP/ADP ratio and increased [Ca²⁺]_i and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP⁺‐induced toxicity. On the other hand, thiol antioxidant N‐acetylcysteine or glutamate receptor antagonist D‐AP5 also partially alleviates MPP⁺‐induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP⁺‐induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μm MPP⁺ induced mitochondrial fragmentation only in tyrosine hydroxylase (TH)‐positive dopaminergic neurons in a similar pattern to that in SH‐SY5Y cells but had no effects on these mitochondrial parameters in TH‐negative neurons. Overall, these findings suggest that DLP1‐dependent mitochondrial fragmentation plays a crucial role in mediating MPP⁺‐induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD.     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.     

NSAIDs protect dopaminergic neurons against 6‐OHDA and MPP+ toxicity

Endogenous and environmental neurotoxins are among the suspected causes of the loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Non‐steroidal anti‐inflammatory drugs (NSAIDs) reduce inflammation by inhibiting cyclooxygenase (COX)‐dependent synthesis of prostaglandins (PG) from arachidonic acid. NSAIDs decrease the incidence of Alzheimer's disease, but little is known about their potential benefit for PD. Therefore, we examined whether NSAIDs could protect DA neurons from neurotoxic insults. NSAIDs can protect DA neurons against excitotoxicity (Casper et al. 2000), and against 6‐hydroxydopamine (6‐OHDA) toxicity (Carrasco et al. 2001). Here, we compared in primary mesencephalic/DA neuron cultures the effect of NSAIDs on the toxicity of 1‐methyl‐phenylpyridinium (MPP⁺) or 6‐OHDA. 6‐OHDA significantly (*p < 0.0001) increased PG production, whereas MPP⁺ did not (p < 0.05). We then compared the competitive/unspecific COX inhibitors ibuprofen and naproxen and the noncompetitive/unspecific inhibitor acetylsalicylic acid (ASA, aspirin) for their ability to protect DA neurons against either 6‐OHDA or MPP⁺ toxicity. Interestingly, all three nonselective COX inhibitors protected DA neurons in cultures against both 6‐OHDA and MPP⁺ (p < 0.05), despite the difference in PG induction by 6‐OHDA vs. MPP⁺. The selective COX‐2 inhibitor NS398 did protect DA neurons against 5 μm MPP⁺ (*p < 0.05), but failed to protect DA neurons against 5 μm 6‐OHDA (p < 0.05). Our results suggest that COX‐inhibitors may have neuroprotective benefits unrelated to inhibition of PG synthesis, and that 6‐OHDA and MPP⁺ have partially overlapping mechanisms of neurodegeneration possibly involving COX activity. Acknowledgement: Supported, in part, by the International Federation for Parkinson's disease, NY, NY.     

Protective Effects of Resveratrol and Quercetin Against MPP<Superscript>+</Superscript> -Induced Oxidative Stress Act by Modulating Markers of Apoptotic Death in Dopaminergic Neurons

Reactive oxygen species produced by oxidative stress may participate in the apoptotic death of dopamine neurons distinctive of Parkinson’s disease. Resveratrol, a red wine extract, and quercetin, found mainly in green tea, are two natural polyphenols, presenting antioxidant properties in a variety of cellular paradigms. The aim of this study was to evaluate the effect of resveratrol and quercetin on the apoptotic cascade induced by the administration of 1-methyl-4-phenylpyridinium ion (MPP⁺), a Parkinsonian toxin, provoking the selective degeneration of dopaminergic neurons. Our results show that a pre-treatment for 3 h with resveratrol or quercetin before MPP⁺ administration could greatly reduce apoptotic neuronal PC12 death induced by MPP⁺. We also demonstrated that resveratrol or quercetin modulates mRNA levels and protein expression of Bax, a pro-apoptotic gene, and Bcl-2, an anti-apoptotic gene. We then evaluated the release of cytochrome c and the nuclear translocation of the apoptosis-inducing factor (AIF). Altogether, our results indicate that resveratrol and quercetin diminish apoptotic neuronal cell death by acting on the expression of pro- and anti-apoptotic genes. These findings support the role of these natural polyphenols in preventive and/or complementary therapies for several human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Nitroindazole compounds inhibit the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to neurotoxic pyridinium cations by human monoamine oxidase (MAO)

Monoamine oxidase (MAO) B is a mitochondrial enzyme selectively involved in the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to toxic pyridinium cations producing Parkinsonism in animal models. Various synthesized 5-nitroindazoles, 6-nitroindazole and the neuroprotectant 7-nitroindazole were examined as inhibitors of MAO and as antioxidants and radical scavengers. The oxidation of MPTP by human MAO-B and mitochondria was assessed by HPLC. Simple nitroindazoles inhibited MPTP oxidation to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) in a competitive and reversible manner. 5-Nitroindazole (IC₅₀=0.99 µM, K_i=0.102 µM) and 6-nitroindazole (IC₅₀=2.5 µM) were better inhibitors of human MAO-B than 7-nitroindazole (IC₅₀=27.8 µM). 6-Nitroindazole also inhibited MAO-A. Nitroindazole isomers were good hydroxyl radical (OH^?) scavengers, with 5-nitro-, 6-nitro- and 7-nitroindazole showing similar activity (k ~10¹⁰ M^?1 s^?1). Neuroprotective actions of nitroindazoles (7-nitroindazole) could be linked to their MAO-inhibitory and antiradical properties besides inhibition on nitric oxide synthase (NOS). 5-Nitro- and 6-nitroindazole, previously reported as weak NOS inhibitors, were better inhibitors of human MAO-B and more active against MPTP neurotoxin oxidation (lower MPDP⁺ and MPP⁺ levels) than 7-nitroindazole and acted as good radical scavengers and could be potential neuroprotective agents in addition to MAO-B inhibitors.     

Neuroprotective Effect of Somatostatin on Nonapoptotic NMDA-Induced Neuronal Death: Role of Cyclic GMP

Abstract: Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca²⁺ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7–10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5–10 µM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25–0.5 mM). GABA (10 µM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 µM) and vapreotide (RC 160; 10 µM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 µM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 µM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5–10 µM). Finally, both specific (Rp-8-bromoguanosine 3′,5′-monophosphate, 10 µM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 µM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca²⁺ level.     

Neurotrophins Rescue Cerebellar Granule Neurons from Oxidative Stress-Mediated Apoptotic Death: Selective Involvement of Phosphatidylinositol 3-Kinase and the Mitogen-Activated Protein Kinase Pathway

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K⁺ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K⁺. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K⁺ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K⁺/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K⁺ to low-K⁺/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K⁺ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1–100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K⁺-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K⁺ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K⁺ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.     

Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP<Superscript>+</Superscript>-induced apoptosis by AKT/GSK-3β/JNK signaling

Parkinson’s disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP⁺ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3β, since inhibition of GSK-3β by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP⁺-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3β inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP⁺-induced apoptotic cell death via PI3K/AKT/GSK-3β pathway, which in turn inhibits MPP⁺-induced JNK activation.