Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining – a non-specific neuronal marker – and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of α-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway,⁶⁵Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When⁶⁵ZnCl₂ was intravenously injected 4 days after treatment with 6-OHDA,⁶⁵Zn was concentrated in the ipsilateral substantia nigra 6 days after⁶⁵Zn injection. On the other hand, 19 d after treatment with 6-OHDA,⁶⁵Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH₄ ⁹⁹TcO₄, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA,⁹⁹Tc was concentrated in the ipsilateral substantia nigra 30 min after⁹⁹Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.     

Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF-mediated signaling

Functions of retinoic acid receptors (RARs) in adult CNS have been poorly characterized. Here we investigated potential neuroprotective action of tamibarotene (Am80), an RARα/β agonist available for the treatment of acute promyelocytic leukemia, on midbrain dopaminergic neurons. Am80 protected dopaminergic neurons in rat midbrain slice culture from injury mediated by lipopolysaccharide-activated microglia, without affecting production of nitric oxide, a key mediator of cell injury. The effect of Am80 was mimicked by another RAR agonist, TAC-101, but not by a retinoid X receptor agonist, HX630, and HX630 did not synergize with Am80. We observed neuronal expression of RARα and RARβ in midbrain slice culture and also found that Am80 increased tissue level of brain-derived neurotrophic factor (BDNF) mRNA. Exogenous BDNF prevented dopaminergic neurodegeneration, and the neuroprotective effect of Am80 was suppressed by a TrkB inhibitor, K252a, or by anti-BDNF neutralizing antibody. These results reveal a novel action of RARs mediated by enhancement of BDNF expression. Finally, oral administration of Am80 prevented dopaminergic cell loss in the substantia nigra induced by local injection of lipopolysaccharide in mice, indicating that RARs are a promising target of therapeutics for neurodegenerative disorders.     

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone,and not mimicked by rh-TNF-alpha,IL-1beta and IFN-gamma

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS-induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti-inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti-inflammatory drug that interferes with many of the features characterizing pro-inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS-injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro-inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS-induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF-alpha/IL-1 beta/IFN-gamma.     

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.     

An immunohistochemical and stereological analysis of PSI-induced nigral neuronal degeneration in the rat

Systemic administration of the proteasomal inhibitor I (PSI) to rats was reported to cause progressive nigral dopaminergic neuronal loss but this is disputed. A major controversy centres over the use of manual counting of tyrosine hydroxylase (TH) positive neurons at the level of third cranial nerve as opposed to employing systematic stereological analysis of cell loss in the entire substantia nigra (SN). To provide a method of marking SN neurones independent of protein expression, fluorogold^™ (FG) was stereotaxically injected bilaterally into the striatum of male Wistar rats to retrogradely label nigral dopaminergic neurons. After 1 week, animals were treated with six doses of PSI (8 mg/kg, s.c.) or its vehicle (dimethyl sulphoxide) on alternate days over a 2-week period. Five weeks after the last treatment, PSI-treated animals showed decreased spontaneous locomotor activity and reduced TH positive SN cell number at the level of the third cranial nerve compared to control rats. Manual cell counting showed loss of FG-labelled SN neurones at this level, with a subpopulation of surviving neurons displaying abnormal morphology. Manual counting of all FG-labelled cells in the entire SN also showed regional PSI-induced loss of neurones with both normal and compromised morphology. Stereological optical fractionator estimates of total FG-labelled cell number confirmed the manual cell counting data both at the level of the third cranial nerve and throughout the entire SN. These findings confirm that PSI does cause a persistent nigral dopaminergic neuronal loss. The reason for the lack of reproducibility between laboratories requires further investigation. We suggest that a failure to distinguish between TH-positive neurones with normal and abnormal morphology following PSI administration contributes to equivocal results.     

Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.     

Role of oxidative stress in paraquat-induced dopaminergic cell degeneration

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time-course of neurodegeneration was correlated to changes in 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4-HNE-positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4-HNE-immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat-induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat-treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4-HNE-positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.     

Protection of dopaminergic neurons by electroconvulsive shock in an animal model of Parkinson's disease

Electroconvulsive shock (ECS) improves motor function in Parkinson's disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6-hydroxydopamine (6-OHDA)-injected rats. Three weeks after a unilateral administration of 6-OHDA, 85–95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6-OHDA-treated animals. Injection of 6-OHDA caused loss of expression of glial cell-line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6-OHDA-treated animals. We also found that protected dopaminergic neurons co-express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6-OHDA induced toxicity.     

A comprehensive study on long‐term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague–Dawley rats. The long‐term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain‐derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase‐positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho‐α‐synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.     

Serofendic acid prevents 6-hydroxydopamine-induced nigral neurodegeneration and drug-induced rotational asymmetry in hemi-parkinsonian rats

Serofendic acid was recently identified as a neuroprotective factor from fetal calf serum. This study was designed to evaluate the neuroprotective effects of an intranigral microinjection of serofendic acid based on behavioral, neurochemical and histochemical studies in hemi-parkinsonian rats using 6-hydroxydopamine (6-OHDA). Rats were injected with 6-OHDA in the presence or absence of serofendic acid, or were treated with serofendic acid on the same lateral side, at 12, 24 or 72 h after 6-OHDA lesion. Intranigral injection of 6-OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta (SNpc). Either simultaneous or 12 h post-administration of serofendic acid significantly prevented both dopaminergic neurodegeneration and drug-induced rotational asymmetry. Immunoreactivities for oxidative stress markers, such as 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE), were markedly detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-administration of serofendic acid, similar to the results in vehicle-treated control rats. In addition, serofendic acid inhibited 6-OHDA-induced alpha-synuclein expression and glial activation in the SNpc. These results suggest that serofendic acid protects against 6-OHDA-induced SNpc dopaminergic neurodegeneration in a rat model of Parkinson's disease.     

A single intramuscular injection of rAAV‐mediated mutant erythropoietin protects against MPTP‐induced parkinsonism

Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson's disease. The constructs were packaged in recombinant adeno‐associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP‐lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7‐ to 9‐Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP‐treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)‐positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4‐dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP‐lesioned mice pretreated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH‐positive fibers in the striatum showed normalized density in MPTP‐lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV‐generated non‐erythropoietic Epo may protect against MPTP‐induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting.     

Spread of neuronal degeneration in a dopaminergic,Lrrk-G2019S model of Parkinson disease

Flies expressing the most common Parkinson disease (PD)-related mutation, LRRK2-G2019S, in their dopaminergic neurons show loss of visual function and degeneration of the retina, including mitochondrial abnormalities, apoptosis and autophagy. Since the photoreceptors that degenerate are not dopaminergic, this demonstrates nonautonomous degeneration, and a spread of pathology. This provides a model consistent with Braak’s hypothesis on progressive PD. The loss of visual function is specific for the G2019S mutation, implying the cause is its increased kinase activity, and is enhanced by increased neuronal activity. These data suggest novel explanations for the variability in animal models of PD. The specificity of visual loss to G2019S, coupled with the differences in neural firing rate, provide an explanation for the variability between people with PD in visual tests.     

Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein

Statins are most commonly prescribed to reduce hypercholesterolemia; however, recent studies have shown that statins have additional benefits, including neuroprotection. Until now, the mechanism underlying statin-induced neuroprotection has been poorly understood. Recent in vivo studies from our lab reported the novel finding that simvastatin increased expression levels of a gene encoding for a major cell survival protein, bcl-2 [Johnson-Anuna et al., J. Pharmacol. Exp. Ther.312 (2005) 786]. The purpose of the present experiments was to determine if simvastatin could protect neurons from excitotoxicity by altering Bcl-2 levels. Neurons were pre-treated with simvastatin and challenged with a compound known to reduce Bcl-2 levels and induce cell death. Simvastatin pre-treatment resulted in a significant reduction in cytotoxicity (lactate dehydrogenase release and caspase 3 activation) following challenge compared with unchallenged neurons. In addition, chronic simvastatin treatment significantly increased Bcl-2 mRNA and protein levels while challenge resulted in a significant reduction in Bcl-2 protein abundance. G3139, an antisense oligonucleotide directed against Bcl-2, abolished the protective effects of simvastatin and eliminated simvastatin-induced up-regulation of Bcl-2 protein. These findings suggest that neuroprotection by simvastatin is dependent on the drug's previously unexplored and important effect of up-regulating Bcl-2.     

Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture

Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.