6-OHDA Toxicity Research Articles

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (≥ 3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Neuroprotective effect of genistein in 6‐hydroxydopamine Hemi‐parkinsonian rat model

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson's disease (PD). Phytoestrogens such as genistein have been reported to prevent neuronal degeneration caused by increased oxidative burden, therefore, this study examined whether genistein administration at a high dose would attenuate behavioral and structural abnormalities in an experimental model of PD in rat. For this purpose, unilateral intrastriatal 6-hydroxydopamine (6-OHDA, 12.5 microg/5 microL of saline-ascorbate)-lesioned rats were intraperitoneally pretreated with a single and high dose of genistein (10 mg/kg) 1 h before surgery. Apomorphine-induced rotations and the number of Nissl-stained neurons in the substantia nigra pars compacta (SNC) were counted after 2 weeks. Genistein administration could attenuate the rotational behavior in lesioned rats and protect the neurons of SNC against 6-OHDA toxicity. Genistein administration has a protective effect against 6-OHDA toxicity.

Time-dependent protective and harmful effects of quercetin on 6-OHDA-induced toxicity in neuronal SH-SY5Y cells

Numerous reports have described the effects of quercetin in models of neurodegenerative diseases, or cancer, resulting in a very complex and sometimes paradoxical picture. Understanding how quercetin causes either protection or cell death in the same model is both tempting and essential. We used the 6-OHDA-induced toxicity model in SH-SY5Y cells, applying graded concentrations of quercetin for a variable time period and following cell viability with MTT-assay and LDH-release as well as caspase-3-like activity. We detected a time-dependent action of quercetin and distinguished an early protective effect from a late toxic one. In addition, we revealed a narrower therapeutic dose-range of quercetin than previously reported in the literature, demonstrating that the toxic effects of quercetin occurred at a concentration only 2-fold higher than the one that produced the greatest protection. We also demonstrated, to our knowledge for the first time, that quercetin itself directly inhibits caspase-3-like activity in a dose-dependent manner. Finally, single doses of quercetin failed to protect against 6-OHDA toxicity in a unilateral rat model of Parkinson's disease. In conclusion, our data may offer an explanation for the dualistic effect of quercetin reported in the literature. In fact, in most studies suggesting quercetin protection against oxidative stressors, the experimental setting failed to include prolonged exposure, and therefore the toxic effects may have been missed. This study supports previous in vivo studies that cast doubt on the efficacy of quercetin against neurodegenerative diseases due to its delayed toxicity.

Neuroprotective effect of genistein in 6-hydroxydopamine hemi-Parkinsonian rat model

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson-s disease (PD). Phytoestrogens such as genistein have been reported to prevent neuronal degeneration caused by increased oxidative burden, therefore, this study examined whether genistein administration at a high dose would attenuate behavioral and structural abnormalities in an experimental model of PD in rat. For this purpose, unilateral intrastriatal 6-hydroxydopamine (6-OHDA, 12.5 μg/5 μL of saline-ascorbate)- lesioned rats were intraperitoneally pretreated with a single and high dose of genistein (10 mg/kg) 1 h before surgery. Apomorphine-induced rotations and the number of Nissl-stained neurons in the substantia nigra pars compacta (SNC) were counted after 2 weeks. Genistein administration could attenuate the rotational behavior in lesioned rats and protect the neurons of SNC against 6-OHDA toxicity. Genistein administration has a protective effect against 6-OHDA toxicity.

Adaptation to chronic MG132 reduces oxidative toxicity by a CuZnSOD‐dependent mechanism

To study whether and how cells adapt to chronic cellular stress, we exposed PC12 cells to the proteasome inhibitor MG132 (0.1 microM) for 2 weeks and longer. This treatment reduced chymotrypsin-like proteasome activity by 47% and was associated with protection against both 6-hydroxydopamine (6-OHDA; 100 microM) and higher dose MG132 (40 microM). Protection developed slowly over the course of the first 2 weeks of exposure and was chronic thereafter. There was no change in total GSH levels after MG132. Buthionine sulfoximine (100 microM) reduced GSH levels by 60%, but exacerbated 6-OHDA toxicity to the same extent in both MG132-treated and control cells and failed to reduce MG132-induced protection. Chronic MG132 resulted in elevated antioxidant proteins CuZn superoxide dismutase (SOD; +55%), MnSOD (+21%), and catalase (+15%), as well as chaperone heat-shock protein 70 (+42%). Examination of SOD enzyme activity revealed higher levels of CuZnSOD (+40%), with no change in MnSOD. We further assessed the mechanism of protection by reducing CuZnSOD levels with two independent siRNA sequences, both of which successfully attenuated protection against 6-OHDA. Previous reports suggested that artificial over-expression of CuZnSOD in dopaminergic cells is protective. Our data complement such observations, revealing that dopaminergic cells are also able to use endogenous CuZnSOD in self-defensive adaptations to chronic stress, and that they can even do so in the face of extensive GSH loss.

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.

Zuckerkandl's organ improves long-term survival and function of neural stem cell derived dopaminergic neurons in Parkinsonian rats

Transplantation of neural stem cells (NSC) derived dopamine (DA) neurons has emerged as an alternative approach to fetal neural cell transplantation in Parkinson's disease (PD). However, similar to fetal neural cell, survival of these neurons following transplantation is also limited due to limited striatal reinnervation (graft with dense neuronal core), limited host–graft interaction, poor axonal outgrowth, lack of continuous neurotrophic factors supply and principally an absence of cell adhesion molecules mediated appropriate developmental cues. In the present study, an attempt has been made to increase survival and function of NSC derived DA neurons, by co-grafting with Zuckerkandl's organ (a paraneural organ that expresses neurotrophic factors as well as cell adhesion molecules); to provide continuous NTF support and developmental cues to transplanted DA neurons in the rat model of PD. 24 weeks post transplantation, a significant number of surviving functional NSC derived DA neurons were observed in the co-transplanted group as evident by an increase in the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, TH-IR fiber density, TH-mRNA expression and TH-protein level at the transplantation site (striatum). Significant behavioral recovery (amphetamine induced stereotypy and locomotor activity) and neurochemical recovery (DA-D2 receptor binding and DA and DOPAC levels at the transplant site) were also observed in the NSC + ZKO co-transplanted group as compared to the NSC or ZKO alone transplanted group. In vivo results were further substantiated by in vitro studies, which suggest that ZKO increases the NSC derived DA neuronal survival, differentiation, DA release and neurite outgrowth as well as protects against 6-OHDA toxicity in co-culture condition. The present study suggests that long-term and continuous NTF support provided by ZKO to the transplanted NSC derived DA neurons, helped in their better survival, axonal arborization and integration with host cells, leading to long-term functional restoration in the rat model of PD.

Leptin Protects against 6-Hydroxydopamine-induced Dopaminergic Cell Death via Mitogen-activated Protein Kinase Signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.

PGE2 receptor EP1 renders dopaminergic neurons selectively vulnerable to low‐level oxidative stress and direct PGE2 neurotoxicity

Oxidative stress and increased cyclooxygenase-2 (COX-2) activity are both implicated in the loss of dopaminergic neurons from the substantia nigra (SN) in idiopathic Parkinson's disease (PD). Prostaglandin E(2) (PGE(2)) is one of the key products of COX-2 activity and PGE(2) production is increased in PD. However, little is known about its role in the selective death of dopaminergic neurons. Previously, we showed that oxidative stress evoked by low concentrations of 6-hydroxydopamine (6-OHDA) was selective for dopaminergic neurons in culture and fully dependent on COX-2 activity. We postulated that this loss was mediated by PGE(2) acting through its receptors, EP1, EP2, EP3, and EP4. Using double-label immunohistochemistry for specific EP receptors and tyrosine hydroxylase (TH), we identified EP1 and EP2 receptors on dopaminergic neurons in rat SN. EP2 receptors were also found in non-dopaminergic neurons of this nucleus, as were EP3 receptors, whereas the EP4 receptor was absent. PGE(2), 16-phenyl tetranor PGE(2) (a stable synthetic analogue), and 17-phenyl trinor PGE(2) (an EP1 receptor-selective agonist) were significantly toxic to dopaminergic cells at nanomolar concentrations; EP2- and EP3-selective agonists were not. We challenged dopaminergic neurons in embryonic rat mesencephalic primary neuronal cultures and tested whether these receptors mediate selective 6-OHDA toxicity. The nonselective EP1-3 receptor antagonist AH-6809 and two selective EP1 antagonists, SC-19220 and SC-51089, completely prevented the 40%-50% loss of dopaminergic neurons caused by exposure to 5 muM 6-OHDA. Together, these results strongly implicate PGE(2) activation of EP1 receptors as a mediator of selective toxicity in this model of dopaminergic cell loss.

Rapid activation of ERK by 6‐hydroxydopamine promotes survival of dopaminergic cells

Isoforms of the mitogen-activated protein kinase ERK have been implicated in both cell survival and cell death. In the present study we explored their role in cell viability in response to oxidative stress. Using the dopaminergic MN9D cell line, we determined that cell death occurred in a concentration-dependent manner after exposure to 6-hydroxydopamine (6-OHDA). The toxicity of 6-OHDA was mediated through generation of reactive oxygen species and was accompanied by a large increase in phosphorylated ERK1/2 but no significant increase in phosphorylated ERK5. 6-OHDA produced a distinct temporal pattern of ERK1/2 activation, with phosphorylated ERK1/2 peaks occurring after 10-15 min (25-fold increase) and 6-24 hr (13-fold increase). Inhibition of the early phosphorylated ERK1/2 peak with U0126 increased the generation of reactive oxygen species by 6-OHDA as well as 6-OHDA-induced toxicity, whereas inhibition of the late peak did not affect 6-OHDA-induced cell death. The time course of phosphorylation of the prosurvival protein CREB mimicked the temporal profile of ERK1/2 activation after 6-OHDA, and blocking the early phospho-ERK1/2 peak also abolished CREB activation. In contrast, activation of caspase-3 by 6-OHDA was delayed, occurring after about 6 hr, and this activation was increased by inhibition of the first phosphorylated ERK1/2 peak. These results suggest that the rapid activation of ERK1/2 in dopaminergic cells by oxidative stress serves as a self-protective response, reducing the content of reactive oxygen species and caspase-3 activity and increasing downstream ERK1/2 substrates.

Myricetin reduces 6-hydroxydopamine-induced dopamine neuron degeneration in rats

The effects of myricetin on 6-hydroxydopamine (6-OHDA)-induced neurodegeneration in the substantia nigra-striatum system were investigated. By high-performance liquid chromatography electrochemical detection, we showed that the dopamine content in the striatum decreased after 6-OHDA treatment, which could be restored by myricetin. The immunohistochemistry and semiquantitative reverse transcription-PCR studies showed that myricetin could prevent the 6-OHDA-induced decrease of tyrosine hydroxylase positive neurons and the tyrosine hydroxylase mRNA expression in the substantia nigra. Perls' iron staining study further demonstrated that myricetin prevented the 6-OHDA-induced increase of iron-staining cells in the substantia nigra. These results suggested that the protective effects of myricetin on the toxicity of 6-OHDA could be attributed to the myricetin-suppressed iron toxicity.

Alpha‐synuclein protects cerebellar granule neurons against 6‐hydroxydopamine‐induced death

The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.

Identification of neuroprotective compounds of Caenorhabditis elegans dopaminergic neurons against 6-OHDA

Parkinson's disease (PD) is a severe debilitating disorder, characterized by progressive and selective dopaminergic (DAergic) neuron degeneration within the substantia nigra pars compacta. Although current pharmacological treatments are effective in early stages of the disease, with time, most patients fail to respond to medications and develop serious motor complications. Therefore, devising novel and efficacious therapeutics that address not only the symptoms of PD, but also the cause, are of great importance. Unfortunately, many obstacles are associated with current PD research in mammalian-based systems, which limit the rate of progress. One solution is to investigate mechanisms of PD in model genetic organisms like Caenorhabditis elegans. In general, striking and profound similarities underlie the basic cellular and molecular processes between the worm and humans. The use of C. elegans over traditional mammalian-based systems holds the promise of an enhanced rate of discovery with lower associated costs. Here, we have utilized C. elegans to screen a variety of compounds, including specific dopamine (DA), GABA, and NMDA receptor agonists, as well as antagonists to identify those that protect against 6-OHDA-induced DAergic toxicity. Two DA D2 receptor agonists, bromocriptine and quinpirole, were found to protect against 6-OHDA toxicity in a dose-dependent manner. Surprisingly, these protective effects appear to involve receptor-independent mechanisms. Given the high degree of conservation of cellular processes between the worm and mammalian systems, these results are likely relevant and important toward understanding potentially novel mechanisms leading to DAergic neuroprotection in mammalian systems and, ultimately, new therapeutics for PD.

6-Hydroxydopamine induces mitochondrial ERK activation

Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 h after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, on initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson's disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury.

Striatal susceptibility to a dopaminergic neurotoxin is independent of sex hormone effects on cell survival and DAT expression but is exacerbated by central aromatase inhibition

The aim of this study was to investigate further the hormone-dependent processes underlying sex differences in neurotoxic responses within the rat nigrostriatal dopaminergic (NSDA) pathway after partial lesioning with 6-OHDA, a state thought to mimic the early stages of Parkinson's disease where, in humans and animal models alike, males appear to be more susceptible. Contrary to our hypotheses, hormone manipulations (gonadectomy +/- oestrogen or androgen treatment) failed to alter survival of tyrosine hydroxylase immunoreactive cells in the substantia nigra pars compacta (SNc) after lesioning; this indicates that, unlike inherent sex differences in toxin-induced striatal dopamine depletion, sex differences in cell loss were not hormonally generated, and that hormone-dependent changes in dopamine depletion can occur independently of cell survival. In addition, hormonally induced changes in striatal expression of the dopamine transporter (DAT), an important factor for 6-OHDA toxicity, did not correlate with hormonal influences on striatal dopamine loss and, in males, central inhibition of aromatase prior to 6-OHDA infusion exacerbated striatal dopamine loss with no effect on SNc tyrosine hydroxylase-immunoreactive survival, suggesting locally generated oestrogen is neuroprotective. These results support the novel view that sex steroid hormones produced peripherally and centrally play a significant, sex-specific role within the sexually dimorphic NSDA pathway to modulate plastic, compensatory responses aimed at restoring striatal dopamine functionality, without affecting cell loss.

A vital role for voltage-dependent potassium channels in dopamine transporter-mediated 6-hydroxydopamine neurotoxicity

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson’s disease models. However, the molecular mechanisms underlying 6-OHDA’s selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)–dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.

Selective Activation of Group III Metabotropic Glutamate Receptors by l-(+)-2-Amino-4-phosphonobutryic Acid Protects the Nigrostriatal System against 6-Hydroxydopamine Toxicity in Vivo

Evidence from several studies suggests that the progressive degeneration of dopaminergic (DA) neurones of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) may in part be due to excessive release of glutamate from subthalamic projections onto nigral DA neurones. Previous in vitro studies have demonstrated that selective activation of Group III metabotropic glutamate receptors (mGluR) negatively modulates excitatory transmission in the SNc and is neuroprotective against glutamate-mediated toxicity. Consistent with this, we have reported preliminary data indicating that the selective group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) can also protect the nigrostriatal system against 6-hydroxydopamine (6-OHDA) toxicity in vivo. We have now extended these preliminary studies in this model and report here that both acute and subchronic intranigral injections of l-AP4 provide significant protection of the nigrostriatal system against 6-OHDA toxicity. This neuroprotection displays a bell-shaped profile with a clear concentration-dependent relationship. In contrast, when administered to animals 7 days post-6-OHDA lesioning, l-AP4 significantly protects the functionality but not the integrity of the nigrostriatal system. We further demonstrate that neuroprotection by l-AP4 in vivo is reversed by coadministration of the selective Group III mGluR antagonist (RS)-alpha-methylserine-O-phosphate, confirming a receptor-mediated mechanism of action. These data provide further compelling evidence that selective activation of Group III mGluR is neuroprotective in an in vivo experimental model of PD, a finding that may have important implications for the future treatment of this disease.

Dopaminergic neuroprotection by neurturin-expressing c17.2 neural stem cells in a rat model of Parkinson's disease

Genetically engineered neural stem cell (NSC) lines are promising vectors for the treatment of regenerative diseases, especially Parkinson's disease (PD). Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor-family, has been demonstrated to act specifically on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for PD. Here, we have generated a NTN-secreting c17.2 NSC line and investigated the protective effect of NTN-c17.2 on PD rat models. These NTN-releasing NSCs engrafted and integrated in the host striatum with good success, gave rise to neurons, astrocytes and oligodendrocytes, and maintained stable, high-level NTN expression. In addition, inverse transfer of NTN protein into the substantia nigra (SN) was able to protect dopaminergic neurons from 6-OHDA toxicity. Observation of rotational behavior showed that the NTN group performed significantly better than the Mock group, and the protective effect of NTN lasted for at least 4 months. HPLC tests indicated that the contents of neurotransmitters (e.g. dopamine) in the corpus striatum area of the NTN-c17.2 group and the Mock-c17.2 group were significantly higher than in the PBS group, but there was no significant difference between expression in the NTN-c17.2 and Mock-c17.2 groups. Taken together, our results suggest that transplantation of NTN-secreting NSCs exerted protective on PD rat models.

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP(+)-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP(+ )or 6-OHDA evoked the elevation of intracellular Ca(2+) levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca(2+) levels due to both toxins. The results show that the MPP(+)-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca(2+) levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca(2+)-independent manner.

Dopamine transporter-mediated cytotoxicity of 6-hydroxydopamine in vitro depends on expression of mutant α-synucleins related to Parkinson's disease

6-Hydroxydopamine (6-OHDA) is widely used to produce animal models of Parkinson's disease (PD) by selectively destroying the nigro-striatal dopaminergic systems, but selective toxicity of 6-OHDA towards dopaminergic cells in vitro remains controversial. Mutant (A30P and A53T) α-synuclein isoforms cause increased vulnerability of cells towards various toxic insults and enhance dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin and mitochondrial complex I inhibitor MPP + in vitro. Here we extend our recent studies on DAT-mediated toxicity to elucidate the mechanisms involved in selective dopaminergic toxicity of 6-OHDA. We studied the cytotoxicity as well as the toxic mechanisms of 6-OHDA in human embryonic kidney HEK-293 cells ectopically co-expressing mutant α-synucleins and the human DAT protein. 6-OHDA showed half-maximal toxic concentration (TC 50) of 88 μM in HEK-hDAT cells without α-synuclein expression after 24 h, whereas the TC 50 values significantly decreased to 58 and 39 μM by expression of A30P and A53T α-synuclein, respectively. α-Synuclein expression did not affect 6-OHDA toxicity in HEK-293 cells not expressing the DAT. Analysis of intracellular parameters of cellular energy metabolism revealed that the co-expression of mutant α-synucleins in HEK-hDAT cells accelerates the reduction of intracellular net ATP levels and ATP/ADP ratios induced by 6-OHDA. Uptake function of the DAT was not altered by expression of α-synuclein isoforms. Our data suggest a mechanism of 6-OHDA-induced dopaminergic toxicity involving an interaction of mutant α-synucleins with the DAT molecule and subsequent acceleration of cellular energy depletion that might be relevant for the pathogenesis of PD.