Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

In order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The nematode Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, provides easy access for molecular studies, has a rapid lifespan, and is an inexpensive model. This study focuses on methods of measuring neurodegeneration involving the dopaminergic system and the identification of compounds with actions that disrupt dopamine function in the model organism C. elegans. Several dopamine-mediated locomotory behaviors, Area Exploration, Body Bends, and Reversals, as well as Swimming-Induced Paralysis and Learned 2-Nonanone Avoidance, were compared to determine the best behavioral method for screening purposes. These behavioral endpoints were also compared to morphological scoring of neurodegeneration in the dopamine neurons. We found that in adult worms, Area Exploration is more advantageous than the other behavioral methods for identifying DA-deficient locomotion and is comparable to neuromorphological scoring outputs. For larval stage worms, locomotion was an unreliable endpoint, and neuronal scoring appeared to be the best method. We compared the wild-type N2 strain to the commonly used dat-1p::GFP reporter strains BY200 and BZ555, and we further characterized the dopamine-deficient strains, cat-2 e1112 and cat-2 n4547. In contrast to published results, we found that the cat-2 strains slowed on food almost as much as N2s. Both showed decreased levels of cat-2 mRNA and DA content, rather than none, with cat-2 e1112 having the greatest reduction in DA content in comparison to N2. Finally, we compared and contrasted strengths, limitations, cost, and equipment needs for all primary methods for analysis of the dopamine system in C. elegans.

Secretome of Undifferentiated Neural Progenitor Cells Induces Histological and Motor Improvements in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that results from the death of dopamine (DA) neurons. Over recent years, differentiated or undifferentiated neural stem cells (NSCs) transplantation has been widely used as a means of cell replacement therapy. However, compelling evidence has brought attention to the array of bioactive molecules produced by stem cells, defined as secretome. As described in the literature, other cell populations have a high-neurotrophic activity, but little is known about NSCs. Moreover, the exploration of the stem cell secretome is only in its initial stages, particularly as applied to neurodegenerative diseases. Thus, we have characterized the secretome of human neural progenitor cells (hNPCs) through proteomic analysis and investigated its effects in a 6-hydroxidopamine (6-OHDA) rat model of PD in comparison with undifferentiated hNPCs transplantation. Results revealed that the injection of hNPCs secretome potentiated the histological recovery of DA neurons when compared to the untreated group 6-OHDA and those transplanted with cells (hNPCs), thereby supporting the functional motor amelioration of 6-OHDA PD animals. Additionally, hNPCs secretome proteomic characterization has revealed that these cells have the capacity to secrete a wide range of important molecules with neuroregulatory actions, which are most likely support the effects observed. Overall, we have concluded that the use of hNPCs secretome partially modulate DA neurons cell survival and ameliorate PD animals' motor deficits, disclosing improved results when compared to cell transplantation approaches, indicating that the secretome itself could represent a route for new therapeutic options for PD regenerative medicine. Stem Cells Translational Medicine 2018;7:829-838.

The protective effects of Citrus aurantium flower extract against 6-hydroxydopamine-mediated cell damage in human neuroblastoma SH-SY5Y cells / Los efectos protectores del extracto de flor de Citrus aurantium contra el daño celular mediado por 6-hidroxidopamina en células humanas de neuroblastoma SH-SY5Y

Parkinson's disease (PD) is described as a neurological condition, resulting from continuous degeneration of dopaminergic neurons. Currently, most treatments for neurodegenerative diseases are palliative. In traditional Iranian medicine, Citrus aurantium flower extract is used to treat some neural diseases, such as sleep disorders and anxiety. The tendency towards the use of medicinal herbs for the treatment of diseases (eg, seizure) is growing. Accordingly, we evaluated the antioxidant effects of C. aurantium flowers and analyzed their protective effects against 6-hydroxydopamine (6-OHDA)-mediated oxidative stress. In this study, 150 mM of 6-OHDA was used to induce cellular damage. Also, MTT assay was performed to analyze cellular viability. Fluorescence spectrophotometry was performed to measure the intracellular reactive oxygen species (ROS) and calcium levels. Based on the findings, 6-OHDA could reduce cell viability. We also analyzed the effects of C. aurantium against neurotoxicity. The intracellular levels of ROS and calcium greatly improved in cells exposed to 6-OHDA. SH-SY5Y cell incubation with C. aurantium (400 and 600 mg/mL) induced protective effects and decreased the biochemical markers of cell apoptosis. According to the findings, C. aurantium showed protective effects against neurotoxicity, caused by 6-OHDA; these protective properties were accompanied by antiapoptotic features. According to the findings, it seems that hydromethanolic C. aurantium extract can be used to prevent seizures.

La enfermedad de Parkinson (EP) se describe como una afección neurológica que resulta de la degeneración continua de las neuronas dopaminérgicas. Actualmente, la mayoría de los tratamientos para las enfermedades neurodegenerativas son paliativos. En la medicina tradicional iraní, el extracto de flor de Citrus aurantium se usa para tratar algunas enfermedades neurológicas, como los trastornos del sueño y la ansiedad. La tendencia hacia el uso de las medicinas para el tratamiento de enfermedades (por ejemplo, convulsiones) está creciendo. Por consiguiente, el objetivo de este trabajo consistió en evaluar los efectos antioxidantes de las flores de C. aurantium y analizar sus efectos protectores contra el estrés oxidativo mediado por la 6- hidroxidopamina (6-OHDA). En este estudio, se usó 150 mM de 6-OHDA para inducir daño celular. Además, se realizó un ensayo de MTT para analizar la viabilidad celular. La espectrofotometría de fluorescencia se realizó para medir las especies reactivas de oxígeno (ROS) intracelulares y los niveles de calcio. En base a los hallazgos, 6-OHDA podría reducir la viabilidad celular. También analizamos los efectos de C. aurantium contra la neurotoxicidad. Los niveles intracelulares de ROS y calcio se expandieron a las células expuestas a 6-OHDA. La incubación de células SH-SY5Y con C. aurantium (400 y 600 mg / ml) indujo efectos protectores y disminuyó los marcadores bioquímicos de la apoptosis celular. De acuerdo con los hallazgos, C. aurantium mostró efectos protectores contra la neurotoxicidad, causada por 6-OHDA; estas propiedades protectoras fueron acompañadas por características antiapoptóticas. Según los hallazgos, parece que el extracto hidrometanólico de C. aurantium se puede usar para prevenir las convulsiones.

Erythropoietin improves neurobehavior by reducing dopaminergic neuron loss in a 6­hydroxydopamine­induced rat model.

The purpose of this study was to determine the effectiveness of the systemic administration of high dose erythropoietin (EPO) in a 6­hydroxydopamine (6­OHDA)­ induced rat model. Rats were divided into 7 groups. Groups 1­4 were administered daily EPO doses of 0; 2,500; 5,000 and 10,000 U/kg via intraperitoneal injection (i.p.) for 5 days. The EPO concentration in cerebrospinal fluid (CSF) was determined by enzyme­linked immunosorbent assay (ELISA) and western blot analysis. The dose of 10,000 U/kg was then selected for subsequent experiments. In group 5, rats received saline via medial forebrain bundle (MFB). In group 6, rats received 6­OHDA via MFB. In group 7, an EPO concentration of 10,000 U/kg was constantly administered i.p. for 5 days to rats prior to 6­OHDA injection via MFB. Behavioral analysis was performed for groups 5­7 by rat rotation tests. The number of tyrosine hydroxylase (TH)­immunopositive cells in the substantia nigra (SN) was measured by immuno-cyto-chemistry. The activation of c­Jun N­terminal kinase (JNK), extracellular signal­regulated kinase (ERK), p38 mitogen­activated protein kinases (MAPKs) and caspase­3 signaling in rats were analyzed using western blotting. The results showed that there was a significant increase in EPO levels in the CSF in 10,000 U/kg group compared with the 2,500 and 5,000 U/kg groups (P<0.01). Significantly fewer rotational counts were obtained in rats that were pretreated with EPO compared with saline­pretreated 6­OHDA­lesioned rats (P<0.001). The dopaminergic neurons in the 6­OHDA­lesioned SN were also increased in the EPO­pretreated rats when compared with control rats (P<0.01). Western blot analysis revealed that EPO inhibited the 6­OHDA­induced activation of JNK, ERK, p38 MAPK and caspase­3 signaling in the rat model. In conclusion, systemic administration of a high dose of EPO exerted neuroprotective effects in reversing behavioral deficits associated with Parkinson's disease and prevented loss of the dopaminergic neurons through the MAPK pathway.

Striatal cholinergic cell ablation attenuates L-DOPA induced dyskinesia in Parkinsonian mice.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia (LID) is a debilitating side effect of long-term dopamine replacement therapy in Parkinson's Disease. At present, there are few therapeutic options for treatment of LID and mechanisms contributing to the development and maintenance of these drug-induced motor complications are not well understood. We have previously shown that pharmacological reduction of cholinergic tone attenuates the expression of LID in parkinsonian mice with established dyskinesia after chronic L-DOPA treatment. The present study was undertaken to provide anatomically specific evidence for the role of striatal cholinergic interneurons by ablating them before initiation of L-DOPA treatment and determining whether it decreases LID. We used a novel approach to ablate striatal cholinergic interneurons (ChIs) via Cre-dependent viral expression of the diphtheria toxin A subunit (DT-A) in hemiparkinsonian transgenic mice expressing Cre recombinase under control of the choline acetyltransferase promoter. We show that Cre recombinase-mediated DT-A ablation selectively eliminated ChIs when injected into striatum. The depletion of ChIs markedly attenuated LID without compromising the therapeutic efficacy of L-DOPA. These results provide evidence that ChIs play a key and selective role in LID and that strategies to reduce striatal cholinergic tone may represent a promising approach to decreasing L-DOPA-induced motor complications in Parkinson's disease.

Neuroprotective effects of swimming training in a mouse model of Parkinson's disease induced by 6-hydroxydopamine.

Parkinson's disease (PD) is characterized by progressive dopamine (DA) depletion in the striatum. Exercise has been shown to be a promising non-pharmacological approach to reduce the risk of neurodegeneration diseases. This study was designed to investigate the potential neuroprotective effect of swimming training (ST) in a mouse model of PD induced by 6-hydroxydopamine (6-OHDA) in mice. The present study demonstrated that a 4-week ST was effective in attenuating the following impairments resulting from 6-OHDA exposure: (i) depressive-like behavior in the tail suspension test; (ii) increase in the number of falls in the rotarod test; (iii) impairment on long-term memory in the object recognition test; (iv) increase of the reactive species and interleukin 1-beta (IL-1ß) levels; (v) inhibition of the glutathione peroxidase (GPx) activity; (vi) rise of the glutathione reductase (GR) and glutathione S-transferase (GST) activities and vii) decrease of DA, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. The mechanisms involved in this study are the modulation of GPx, GR and GST activities as well as IL-1ß level in a PD model induced by 6-OHDA, protecting against the decrease of DA, DOPAC and HVA levels in the striatum of mice. These findings reinforce that one of the effects induced by exercise on neurodegenerative disease, such as PD, is due to antioxidant and anti-inflammatory properties. We suggest that exercise attenuates cognitive and motor declines, depression, oxidative stress, and neuroinflammation induced by 6-OHDA supporting the hypothesis that exercise can be used as a non-pharmacological tool to reduce the symptoms of PD.

One-electron reduction of 6-hydroxydopamine quinone is essential in 6-hydroxydopamine neurotoxicity.

6-Hydroxydamine has widely been used as neurotoxin in preclinical studies related on the neurodegenerative process of dopaminergic neurons in Parkinson's disease based on its ability to be neurotoxic as a consequence of free radical formation during its auto-oxidation to topaminequinone. We report that 50-µM 6-hydroxydopamine is not neurotoxic in RCSN-3 cells derived from substantia nigra incubated during 24 h contrasting with a significant sixfold increase in cell death (16 ± 2 %; P < 0.001) was observed in RCSN-3NQ7 cells expressing a siRNA against DT-diaphorase that silence the enzyme expression. To observe a significant cell death in RCSN-3 cells induced by 6-hydroxydopamine (24 ± 1 %; P < 0.01), we have to increase the concentration to 250 µm while a 45 ± 2 % cell death (P < 0.001) was observed at this concentration in RCSN-3NQ7 cells. The cell death induced by 6-hydroxydopamine in RCSN-3NQ7 cells was accompanied with a (i) significant increase in oxygen consumption (P < 0.01), (ii) depletion of reduced glutathione and (iii) a significant decrease in ATP level (P < 0.05) in comparison with RCSN-3 cells. In conclusion, our results suggest that one-electron reduction of 6-hydroxydopamine quinone seems to be the main reaction responsible for 6-hydroxydopamine neurotoxic effects in dopaminergic neurons and DT-diaphorase seems to play an important neuroprotective role by preventing one-electron reduction of topaminequinone.

Research on an in vitro cell system for testing the neurotoxicity of kynurenine pathway metabolites.

Quinolinic acid (QUIN), kynurenine acid (KYNA) and 3-hydroxykynurenine (3-HK) - metabolites of the kynurenine pathway are considered to be associated with many central nervous system diseases. However, in neuroscience research in order to test neurotoxicity or neuroprotection against these compounds only primary cell models are available. In this investigation we aimed to develop a simple, rapid and accurate cellular in vitro model using immortalized human neuroblastoma cell lines, namely SK-N-SH and SH-SY5Y differentiated by treatment with various agents. In order to alter the cell response to the neurotoxins, tumor necrosis factor-alpha and retinoic acid (RA) as differentiating agents and modulation of the cellular metabolism through changing the sugar composition from galactose to glucose in media were used. Our results indicated that although RA-differentiation of both cell lines induced the expression of neuronal features, cell vulnerability after exposure to control neurotoxicants (salsolinol, 6-hydroxydopamine) and 3-HK was decreased in comparison to untreated cells and was not influenced after exposure to QUIN and KYNA. Interestingly, the same observations were done in cells grown in galactose containing media.

Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons.

Pitx3 is a critical homeodomain transcription factor for the proper development and survival of mesodiencephalic dopaminergic (mdDA) neurons in mammals. Several variants of this gene have been associated with human Parkinson's disease (PD), and lack of Pitx3 in mice causes the preferential loss of substantia nigra pars compacta (SNc) mdDA neurons that are most affected in PD. It is currently unclear how Pitx3 activity promotes the survival of SNc mdDA neurons and which factors act upstream and downstream of Pitx3 in this context. Here we show that a transient expression of glial cell line-derived neurotrophic factor (GDNF) in the murine ventral midbrain (VM) induces transcription of Pitx3 via NF-κB-mediated signaling, and that Pitx3 is in turn required for activating the expression of brain-derived neurotrophic factor (BDNF) in a rostrolateral (SNc) mdDA neuron subpopulation during embryogenesis. The loss of BDNF expression correlates with the increased apoptotic cell death of this mdDA neuronal subpopulation in Pitx3(-/-) mice, whereas treatment of VM cell cultures with BDNF augments the survival of the Pitx3(-/-) mdDA neurons. Most importantly, only BDNF but not GDNF protects mdDA neurons against 6-hydroxydopamine-induced cell death in the absence of Pitx3. As the feedforward regulation of GDNF, Pitx3, and BDNF expression also persists in the adult rodent brain, our data suggest that the disruption of the regulatory interaction between these three factors contributes to the loss of mdDA neurons in Pitx3(-/-) mutant mice and perhaps also in human PD.

Amantadine attenuates levodopa-induced dyskinesia in mice and rats preventing the accompanying rise in nigral GABA levels.

Amantadine is the only drug marketed for treating levodopa-induced dyskinesia. However, its impact on basal ganglia circuitry in the dyskinetic brain, particularly on the activity of striatofugal pathways, has not been evaluated. We therefore used dual probe microdialysis to investigate the effect of amantadine on behavioral and neurochemical changes in the globus pallidus and substantia nigra reticulata of 6-hydroxydopamine hemi-lesioned dyskinetic mice and rats. Levodopa evoked abnormal involuntary movements (AIMs) in dyskinetic mice, and simultaneously elevated GABA release in substantia nigra reticulata (∼3-fold) but not globus pallidus. Glutamate levels were unaffected in both areas. Amantadine (40 mg/kg, i.p.), ineffective alone, attenuated (∼50%) AIMs expression and prevented the GABA rise. Moreover, it unraveled a facilitatory effect of levodopa on pallidal glutamate levels. Levodopa also evoked AIMs expression and a GABA surge (∼2-fold) selectively in the substantia nigra of dyskinetic rats. However, different from mice, glutamate levels rose simultaneously. Amantadine, ineffective alone, attenuated (∼50%) AIMs expression preventing amino acid increase and leaving unaffected pallidal glutamate. Overall, the data provide neurochemical evidence that levodopa-induced dyskinesia is accompanied by activation of the striato-nigral pathway in both mice and rats, and that the anti-dyskinetic effect of amantadine partly relies on the modulation of this pathway.

Synergy between glutathione peroxidase-1 and astrocytic growth factors suppresses free radical generation and protects dopaminergic neurons against 6-hydroxydopamine.

The degeneration of dopaminergic neurons in the course of Parkinson disease is largely blamed on oxidative damage in the brain. This study examined the potency of glutathione peroxidase-1 (GPX-1) to protect dopaminergic neurons against toxicity induced by the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). We generated pLV-GPX1, a recombinant lentivirus vector carrying the coding sequence for human GPX-1, into the SK-N-MC neuroblastoma cell line. The pLV-GPX1-infected neurons showed an over 3-fold increase in enzyme expression and a 2.6-fold increase in enzyme activity compared to the pLV-EGFP-infected control cells. In the pLV-GPX1-infected cells, we also detected significantly increased neuronal survival and resistance to 6-OHDA-mediated toxicity compared to our controls (75 ± 4% versus 51 ± 7%, p < 0.001). To maximize this protection, the neurons were treated with conditioned medium taken from growing primary astrocytes (astro-CM). We found the treated pLV-GPX1-infected neurons even more significantly resistant to 6-OHDA toxicity compared to their untreated counterparts (86 ± 5% versus 75 ± 4%, p < 0.001). Concomitant with increased neuroprotection, co-presence of overexpressed GPX-1 and astro-CM significantly increased glutathione (GSH) levels compared to when either of the two was present (p < 0.001). Further analysis showed nearly 2.7-fold reduction, in the presence of astro-CM, of hydrogen peroxide (H(2)O(2)) levels released from the pLV-GPX1-infected neurons compared to control groups (p < 0.001). Finally, regression analysis between H(2)O(2) levels and cell viability showed that co-presence of GPX-1 and astro-CM reduced 33% of cell death rate (p < 0.05). These data highlight the antioxidant properties of GPX-1 in protecting dopaminergic neurons and further emphasize the capacity of astrocytes in pumping growth-inducing factors that may synergize with GPX-1 to accelerate neuroprotection.

[Protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats].

OBJECTIVE: To investigate the protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats. METHODS: Thirty male SD rats were divided into four groups. Uric acid of 100 mg/kg, 200 mg/kg, 250 mg/kg were injected intraperitoneally (ip) into 5, 10, 5 rats twice daily at a 2-hour interval for five days and saline was injected ip into 10 rats as controls. At Day 6, 6-hydroxydopamine was injected into striatum to establish Parkinson's disease (PD) model in rats. Then uric acid was injected ip into three groups and saline into controls for five days. Locomotion test, amphetamine-induced rotation and forepaw adjusting step test were performed at Weeks 3 and 4 respectively after injection of 6-hydroxydopamine. HPLC-MS/MS was performed to detect the contents of dopamine and its metabolite homovanillic acid (HVA) in striatum at Week 5. RESULTS: The scores of locomotion in 2 minutes of 200 mg/kg uric acid group (14 +/- 4/2 min) was higher significantly than saline group (4 +/- 5/2 min, P < 0.01). The amphetamine-induced rotation number in the 200 mg/kg uric acid group (10.8 +/- 7.5) was lower significantly that in the saline group (19.3 +/- 5.2, P < 0.01). Forepaw adjusting step test scores of 200 mg/kg uric acid group were higher significantly than those in the saline group (9.89 +/- 3.41 vs 4.36 +/- 3.72, P < 0.01). HPLC-MS/MS showed that the contents of DA (0.29 +/- 0.19) and HVA (1.22 +/- 0.5) in injured striatum of 200 mg/kg uric acid group were higher significantly than those in the saline group (0.05 +/- 0.03, P < 0.01; 0.24 +/- 0.13, P < 0.05). CONCLUSION: An appropriately elevated level of uric acid may protect the dopamine neuron of nigrostriatal system from injury of 6-hydroxydopamine in rats.

Puerarin protects dopaminergic neurons against 6-hydroxydopamine neurotoxicity via inhibiting apoptosis and upregulating glial cell line-derived neurotrophic factor in a rat model of Parkinson's disease.

Neurodegeneration is one of the primary etiologies in the onset of Parkinson's disease. In the quest for a new antiparkinsonism treatment the potential benefits of puerarin from the roots of Pueraria lobata have been recognized. Thus, we examined whether puerarin is capable to protect dopaminergic neurons of the substantia nigra against 6-hydroxydopamine induced neuronal cell death. Our data showed that the intraperitoneal administration of 0.12 mg/kg/day puerarin over 10 days reduced the 6-hydroxydopamine-induced decrease of tyrosine hydroxylase-positive cell counts. Analysis of apoptosis via DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay proved that puerarin could prevent 6-hydroxydopamine-induced apoptosis. As an additional apoptotic cell death marker, the BAX and BCL-2 expression levels were investigated using immunohistochemistry. Whereas 6-hydroxydopamine increased the level of Bax (p < 0.05), the coadministrated puerarin significantly antagonized this effect in a dose-dependent manner. Bcl-2 expression was not affected. Analysis of the dopamine, dihydroxyphenylacetic acid, and L-dihydroxy-phenyl-alanine contents of 6-hydroxydopamine-treated animals by HPLC revealed that puerarin was capable to restore the contents of dopamine and its metabolites. Moreover, the expression level of glial cell line-derived neurotrophic factor in the striatum was higher in puerarin than in rats treated with 6-hydroxydopamine alone. These results suggest that puerarin develops its neuroprotective effect against 6-hydroxydopamine-induced neurotoxicity in the substantia nigra through the inhibition of apoptotic signaling pathways and upregulation of glial cell line-derived neurotrophic factor expression in the striatum.

Up-regulation of divalent metal transporter 1 in 6-hydroxydopamine intoxication is IRE/IRP dependent.

Iron plays a key role in Parkinson's disease (PD). Increased iron content of the substantia nigra (SN) has been found in PD patients, and divalent metal transporter 1 (DMT1) has been shown to be up-regulated in the SN of both MPTP-induced PD models and PD patients. However, the mechanisms underlying DMT1 up-regulation are largely unknown. In the present study, we observed that in the SN of 6-hydroxydopamine (6-OHDA)-induced PD rats, DMT1 with the iron responsive element (IRE, DMT1+IRE), but not DMT1 without IRE (DMT1-IRE), was up-regulated, suggesting that increased DMT1+IRE expression might account for nigral iron accumulation in PD rats. This possibility was further assessed in an in vitro study using 6-OHDA-treated and DMT1+IRE-over-expressing MES23.5 cells. In 6-OHDA-treated MES23.5 cells, increased iron regulatory protein (IRP) 1 and IRP2 expression was observed, while silencing of IRPs dramatically diminished 6-OHDA-induced DMT1+IRE up-regulation. Pretreatment with N-acetyl-L-cysteine fully suppressed IRPs up-regulation by inhibition of 6-OHDA-induced oxidative stress. Increased DMT1+IRE expression resulted in increased iron influx by MES23.5 cells. Our data provide direct evidence that DMT1+IRE up-regulation can account for IRE/IRP-dependent 6-OHDA-induced iron accumulation initiated by 6-OHDA-induced intracellular oxidative stress and that increased levels of intracellular iron result in aggravated oxidative stress. The results of this study provide novel evidence supporting the use of anti-oxidants in the treatment of PD, with the goal of inhibiting iron accumulation by regulation of DMT1 expression.

The Wld(S) mutation delays anterograde, but not retrograde, axonal degeneration of the dopaminergic nigro-striatal pathway in vivo.

For many neurodegenerative disorders, such as Parkinson's disease, there is evidence that the disease first affects axons and terminals of neurons that are selectively vulnerable. This would suggest that it may be possible to forestall progression by targeting the cellular mechanisms of axon degeneration. While it is now clear that these mechanisms are distinct from the pathways of programmed cell death, they are less well known. Compelling evidence of the distinctiveness of these mechanisms has derived from studies of the Wld(S) mutation, which confers resistance to axon degeneration. Little is known about how this mutation affects degeneration in dopaminergic axons, those that are affected in Parkinson's disease. We have characterized the Wld(S) phenotype in these axons in four models of injury: two that utilize the neurotoxin 6-hydroxydopamine or axotomy to induce anterograde degeneration, and two that use these methods to induce retrograde degeneration. For both 6-hydroxydopamine and axotomy, Wld(S) provides protection from anterograde, but not retrograde degeneration. This protection is observed as preserved immunostaining for tyrosine hydroxylase in axons and striatum, and by structural integrity visualized by GFP in tyrosine hydroxylase-GFP mice. Therefore, Wld(S) offers axon protection, but it reveals fundamentally different processes underlying antero- and retrograde degeneration in this system.

Fetal striatum- and ventral mesencephalon-derived expanded neurospheres rescue dopaminergic neurons in vitro and the nigro-striatal system in vivo.

The pathogenesis of Parkinson's disease (PD) involves ongoing apoptotic loss of dopaminergic neurons in the substantia nigra pars compacta. Local delivery of the trophic factors can rescue dopaminergic neurons and halt the progression of PD. In this study we show that fetal E11 striatum-derived neurospheres and E14.5 ventral mesencephalon (VM) -derived neurospheres (NS E11 and NSvm, respectively) are a source of factors that rescue dopaminergic neurons. First, long-term expanded NS E11 and NSvm rescued primary dopaminergic neurons from serum-deprivation induced apoptosis and promoted survival of dopaminergic neurons for 14 days in vitro and this effect was due to soluble contact-independent factor/s. Second, green fluorescent protein-expressing NS E11 and NSvm grafted into the midbrain of mice with unilateral 6-hydroxydopamine-induced Parkinsonism resulted in partial rescue of the nigro-striatal system and improvement of the hypo-dopaminergic behavioral deficit. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that intact NS E11 and NSvm expressed fibroblast growth factor-2, brain-derived neurotrophic factor (BDNF), pleiotrophin, neurotrophin-3, but not glial cell line-derived neurotrophic factor (GDNF). GDNF expression was also undetectable in vivo in grafted NS E11 and NSvm suggesting that NS-derived factor/s other than GDNF mediated the rescue of nigral dopaminergic neurons. Identification of NS-derived soluble factor(s) may lead to development of novel neuroprotective therapies for PD. An unexpected observation of the present study was the detection of the ectopic host-derived tyrosine hydroxylase (TH) -expressing cells in sham-grafted mice and NS E11- and NSvm -grafted mice. We speculate that injury-derived signals (such as inflammatory cytokines that are commonly released during transplantation) induce TH expression in susceptible cells.

Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson's disease.

Both fetal ventral mesencephalic (VM) and embryonic stem (ES) cell-derived dopamine neurons have been used successfully to correct behavioral responses in animal models of Parkinson's disease. However, grafts derived from fetal VM cells or from ES cells contain multiple cell types, and the majority of these cells are not dopamine neurons. Isolation of ES cell-derived dopamine neurons and subsequent transplantation would both elucidate the capacity of these neurons to provide functional input and also further explore an efficient and safer use of ES cells for the treatment of Parkinson's disease. Toward this goal, we used a Pitx3-enhanced green fluorescent protein (Pitx3-eGFP) knock-in mouse blastocyst-derived embryonic stem (mES) cell line and fluorescence-activated cell sorting (FACS) to select and purify midbrain dopamine neurons. Initially, the dopaminergic marker profile of intact Pitx3-eGFP mES cultures was evaluated after differentiation in vitro. eGFP expression overlapped closely with that of Pitx3, Nurr1, Engrailed-1, Lmx1a, tyrosine hydroxylase (TH), l-aromatic amino acid decarboxylase (AADC), and vesicular monoamine transporter 2 (VMAT2), demonstrating that these cells were of a midbrain dopamine neuron character. Furthermore, postmitotic Pitx3-eGFP(+) dopamine neurons, which constituted 2%-5% of all live cells in the culture after dissociation, could be highly enriched to >90% purity by FACS, and these isolated neurons were viable, extended neurites, and maintained a dopaminergic profile in vitro. Transplantation to 6-hydroxydopamine-lesioned rats showed that an enriched dopaminergic population could survive and restore both amphetamine- and apomorphine-induced functions, and the grafts contained large numbers of midbrain dopamine neurons, which innervated the host striatum. Disclosure of potential conflicts of interest is found at the end of this article.

Effect of selank on cognitive processes after damage inflicted to the cerebral catecholamine system during early ontogeny.

Effects of selank on learning, memory, and attention to sensory stimuli of different modality were studied in adult Wistar rats treated with 6-hydroxydopamine (neurotoxin selectively damaging catecholaminergic neurons and their terminals) during the first 3 days of life. Selank (300 microg/kg) restored cognitive processes disordered by chronic artificial inhibition of the cerebral catecholaminergic system.

Differential involvement of mitochondrial permeability transition in cytotoxicity of 1-methyl-4-phenylpyridinium and 6-hydroxydopamine.

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the influence of the mitochondrial membrane permeability transition inhibition against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in relation to the mitochondria-mediated cell death process and role of oxidative stress. Both MPP(+) and 6-OHDA induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Cyclosporin A (CsA), trifluoperazine and aristolochic acid, inhibitors of mitochondrial permeability transition, significantly attenuated the MPP(+)-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. In contrast to MPP(+), the cytotoxicity of 6-OHDA was not reduced by the addition of the mitochondrial permeability transition inhibitors. The results show that the cytotoxicity of MPP(+) may be mediated by the mitochondrial permeability transition formation, which is associated with formation of reactive oxygen species and the depletion of GSH. In contrast, the 6-OHDA-induced cell injury appears to be mediated by increased oxidative stress without intervention of the mitochondrial membrane permeability transition.