l-Deprenyl Protects Mesencephalic Dopamine Neurons from Glutamate Receptor-Mediated Toxicity In Vitro

Abstract: l -Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)-B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO-B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µ M ) and the NMDA receptor blocker MK-801 (10 µ M ) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µ M ) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µ M ) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.     

FK506 Protects Neurons Following Peripheral Nerve Injury Via Immunosuppression

In this study, we have evaluated neuroprotective effect of an immunosuppressant immunophilin ligand, FK506, in the sciatic nerve injury model in rats. FK506 was injected to the sciatic nerve transected 3-month-old female Wistar rats (2 mg/kg/day starting 1 day prior to sciatic nerve injury up to 7 day post operation). Equal number of sciatic nerve transected animals served as injured untreated controls. The contralateral side served as respective control. L4-L5 region of the spinal cord was removed on day 1, 3, 7, 14, 21, and 28, post operation and then processed for cryo-sectioning and paraffin sectioning. The cryocut sections were used for immunohistochemistry for localizing all microglia (using anti-Iba-1) and MHC-II expressing microglia (with OX-6). The physical dissector method was applied on Nissl stained paraffin sections for absolute motor neuron counting in the L4-L5 region of spinal cord. FK506 treated animals presented 88.7% neuronal survival while the injured alone had 79.12%, which is significantly less than the treated animals. FK506 caused early proliferation of microglia at 1 and 3 days post operation. FK506 also significantly restricted transformation of these cells in to phagocytes. Colocalization of activated microglia by anti-Iba-1 and OX-6 antibodies, confirms that the MHC-II expressing cells in injured spinal cord are none other than microglial cells and MHC-II expressing cells are significantly less in treated as compared to untreated injured animals. We propose that immunosuppression is one of the main mechanisms by which FK506 protects the central neurons following peripheral injury.     

Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons

Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate-induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate-induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate-induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.     

A Simplified Method for Isolating Highly Purified Neurons, Oligodendrocytes, Astrocytes, and Microglia from the Same Human Fetal Brain Tissue

Elucidation of the underlying pathogenic mechanisms leading to apoptosis of neurons and oligodendrocytes and activation of microglia and astrocytes in different neurodegenerative and neuroinflammatory disorders remains a challenge in neuroscience. In order to overcome the challenge and find out therapeutic remedies, it is important to study live and death processes in each and every cell type of the brain. Here we present a protocol of isolating highly purified microglia, astrocytes, oligodendrocytes, and neurons, all four major cell types of the CNS, from the same human fetal brain tissue. As found in vivo, these primary neurons and oligodendroglia underwent apoptosis and cell death in response to neurodegenerative challenges. On the other hand, astroglia, and microglia, cells that do not die in neurodegenerative brains, became activated after inflammatory challenge. The availability of highly purified human brain cells will increase the possibility of developing therapies for different neurodegenerative disorders. M. Jana and A. Jana have equal contribution to the work.     

Inflammatory Mediator Stimulation of Astrocytes and Meningeal Fibroblasts Induces Neuronal Degeneration via the Nitridergic Pathway

Abstract: The role of inflammatory cytokines in the pathogenesis of neurological disorders is not entirely clear. The neurotoxic effects of cytokines, and perhaps indirectly bacterial endotoxins, could be mediated by the stimulation of immunocompetent cells in the brain to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. NO is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. Both constitutive and inducible NO synthase has been described in astrocytes in vitro. Here we demonstrate that newborn mouse cortical astrocytes, when coincubated with neonatal mouse cerebellar granule cells or hippocampal neurons, induced neurotoxicity upon stimulation with endotoxin (lipopolysaccharide) (ED₅₀ 30 ng/ml). Astrocytes were unresponsive to the cytokines tumor necrosis factor-α or interleukin-1β individually, but exhibited a marked synergistic stimulation in their combined presence. Moreover, meningeal fibroblasts treated with tumor necrosis factor-α, but not interleukin-1β or lipopolysaccharide, elaborated neurotoxicity for cocultured granule cells (ED₅₀ 30 U/ml). In cocultures of immunostimulated astrocytes or meningeal fibroblasts, neurotoxicity was blocked by the NO synthase inhibitors N^ω-nitro-l -arginine and N^ω-nitro-d -arginine methyl ester, and by oxyhemoglobin, which inactivates NO. Astroglial-induced neurotoxicity was not affected by N-methyl-d -aspartate receptor antagonists. Superoxide dismutase, which degrades superoxide anion, attenuated astrocyte- and fibroblast-mediated neurotoxicity, indicating that endogenous superoxide anion may react with NO to form toxic peroxynitrite and its breakdown products. These findings suggest a potentially important role for glial- and meningeal fibroblast-induced NO synthase in the pathophysiology of CNS disease states of immune or inflammatory origin.     

Neuron-derived IgG Protects Neurons from Complement-dependent Cytotoxicity

Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.     

Protective Actions of the Vesicular Monoamine Transporter 2 (VMAT2) in Monoaminergic Neurons

Vesicular monoamine transporters (VMATs) are responsible for the packaging of neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine into synaptic vesicles. These proteins evolved from precursors in the major facilitator superfamily of transporters and are among the members of the toxin extruding antiporter family. While the primary function of VMATs is to sequester neurotransmitters within vesicles, they can also translocate toxicants away from cytosolic sites of action. In the case of dopamine, this dual role of VMAT2 is combined—dopamine is more readily oxidized in the cytosol where it can cause oxidative stress so packaging into vesicles serves two purposes: neurotransmission and neuroprotection. Furthermore, the deleterious effects of exogenous toxicants on dopamine neurons, such as MPTP, can be attenuated by VMAT2 activity. The active metabolite of MPTP can be kept within vesicles and prevented from disrupting mitochondrial function thereby sparing the dopamine neuron. The highly addictive drug methamphetamine is also neurotoxic to dopamine neurons by using dopamine itself to destroy the axon terminals. Methamphetamine interferes with vesicular sequestration and increases the production of dopamine, escalating the amount in the cytosol and leading to oxidative damage of terminal components. Vesicular transport seems to resist this process by sequestering much of the excess dopamine, which is illustrated by the enhanced methamphetamine neurotoxicity in VMAT2-deficient mice. It is increasingly evident that VMAT2 provides neuroprotection from both endogenous and exogenous toxicants and that while VMAT2 has been adapted by eukaryotes for synaptic transmission, it is derived from phylogenetically ancient proteins that originally evolved for the purpose of cellular protection.     

l-(−)-Desmethylselegiline, a Metabolite of Selegiline [l-(−)-Deprenyl], Protects Mesencephalic Dopamine Neurons from Excitotoxicity In Vitro

Abstract: Selegiline [ l -(−)-deprenyl], a monoamine oxidase B inhibitor, has been used in the treatment of Parkinson's disease as a putative neuroprotective agent. Selegiline is metabolized rapidly in the gastrointestinal tract and liver to desmethylselegiline (DMS) and methamphetamine. We have previously shown that selegiline protects dopamine neurons in mesencephalic cultures from toxicity resulting from activation of glutamate receptors. In the present study we examined whether DMS has similar neuroprotective effects. Our data show that DMS protects dopamine neurons from N -methyl- d -aspartate receptor-mediated excitotoxic damage. The efficacy of DMS is greater than that of selegiline, as it can cause protection at lower concentrations and provide significantly greater levels of protection at the same concentrations. Our results suggest that DMS might be the active compound responsible for the neuroprotective properties of selegiline.     

The Desglycinyl Metabolite of Remacemide Hydrochloride Is Neuroprotective in Cultured Rat Cortical Neurons

Abstract: The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR), a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 µ M ) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5–20 µ M ), but not its remacemide precursor (10–100 µ M ), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5–20 µ M ) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 µ M NMDA and reduced the NMDA-triggered increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by up to 70%. By contrast, remacemide (50 and 100 µ M ) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca²⁺]_i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca²⁺]_i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca²⁺]_i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.     

Catalpol protects dopaminergic neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Microglia, the resident immune cells in the central nervous system, are pivotal in the inflammatory reaction. Activated microglia can induce expression of inducible nitric-oxide synthase (iNOS) and release significant amounts of nitric oxide (NO) and TNF-alpha, which can damage the dopaminergic neurons. Catalpol, an iridoid glycoside, contained richly in the roots of Rehmannia glutinosa, was found to be neuroprotective in gerbils subjected to transient global cerebral ischemia. But the effect of catalpol on inflammation-mediated neurodegeneration has not been examined. In this study, microglia in mesencephalic neuron-glia cultures were activated with lipopolysaccharide (LPS) and the aim of the study was to examine whether catalpol could protect dopaminergic neurons from LPS-induced neurotoxicity. The results showed that catalpol significantly reduced the release of reactive oxygen species (ROS), TNF-alpha and NO after LPS-induced microglial activation. Further, catalpol attenuated LPS-induced the expression of iNOS. As determined by immunocytochemical analysis, pretreatment by catalpol dose-dependently protected dopaminergic neurons against LPS-induced neurotoxicity. These results suggest that catalpol exerts its protective effect on dopaminergic neurons by inhibiting microglial activation and reducing the production of proinflammatory factors. Thus, catalpol may possess therapeutic potential against inflammation-related neurodegenerative diseases.     

Ubiquinone-10 plasma concentrations in healthy European children

The reduced form of ubiquinone-10 (coenzyme Q) has been shown to represent an important physiologic antioxidant principle in human blood. In order to establish a reference range for infants, we measured plasma levels of ubiquinone in 50 healthy European children aged 2 months to 15 years. A mean ±SD) value of 0.75±0.27 μg/ml plasma (0.87±0.31 μM) was determined; ubiquinone concentrations were not found to be sex-dependent (0.7±0.24μg/ml for girls, n=17, and 0.7±0.28μg/ml for boys, n=33) but correlated negatively with age (r = -0.37, P=0.0075). This negative correlation was mainly due to relatively high levels in infants approximately 1 year old.

The mean value determined does not significantly differ from the average ubiquinone plasma concentrations determined in healthy Nigerian children (0.85±0.40 μg/ml, n= 18) in a previous study (Becker K, Boetticher D, Leichsenring M. Internat J Vitam Nutr Res 1995, in press).     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Adenosine Transport by Primary Cultures of Neurons from Chick Embryo Brain

Abstract: The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [³H]adenosine. During the 5-25-s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis-Menten relationship and yielded a K_m of 13 μM and V_max of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10-100-fold higher than [³H]adenosine. Adenosine derivatives ( N ⁶-cyclohexyl-, N⁶-benzyl-, N⁶-methyl-, and 2-chloroadenosine) were more effective inhibitors; p -nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high-affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.     

N-Methyl-d-Aspartate- or Glutamate-Mediated Toxicity in Cultured Rat Cortical Neurons Is Antagonized by FPL 15896AR

Abstract: The neuroprotective action of ( S )-α-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N -methyl- d -aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 µ M ) or glutamate (50 µ M ) for 15 min resulted in the death of 85–95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 µ M ) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 µ M ) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 µ M NMDA or 50 µ M glutamate. FPL 15896AR also reduced by ∼35% the magnitude of NMDA-triggered increases in intracellular free Ca²⁺ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.     

Aurintricarboxylic Acid Protects Hippocampal Neurons from Glutamate Excitotoxicity In Vitro

Abstract: Aurintricarboxylic acid (ATA), an endonuclease inhibitor, has been shown to protect several cell types from an apoptotic form of cell death. We tested ATA for protective effects against glutamate excitotoxicity in 2-week-old cultured hippocampal neurons. Cell viability was determined 24 h after glutamate exposure either by trypan blue exclusion or by measurement of lactate dehydrogenase release. When ATA was added during exposure to glutamate, there was a dramatic increase in the number of viable neurons compared with cultures that did not receive ATA. If ATA was added after glutamate exposure, the rate of survival approached 100%. Several cellular processes may be the targets for ATA action. If the mechanisms of ATA protection are similar for excitotoxicity and apoptosis, then these distinct forms of cell death may share a common intracellular pathway.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation

Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.