Pharmacoprotective influences on different links of the mechanism underlying 6-hydroxydopamine-induced degeneration of nigro-striatal dopaminergic neurons

In this study we examined the possibility of preventing degeneration of dopaminergic nigro-striatal neurons caused in rats by a neurotoxin, 6-hydroxydopamine. We showed that an antioxidant, Trolox, an inhibitor of nitric oxide synthase, N_ω-nitro-L-arginine methyl ester, and an inhibitor of caspases, zinc chloride, are capable of preventing to a considerable extent the neurotoxin-induced apoptosis of dopaminergic neurons of the substantia nigra. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 150–156, March–April, 2006.     

Reconstruction of dopaminergic neural network and locomotion function in planarian regenerates

Planarian, an invertebrate flatworm, has a high capacity for regeneration when compared with other worms and animals. We show here for the first time that the reconstructed dopamine (DA) neural network regulates locomotion and behavior in planarian regenerates. The gene encoding tyrosine hydroxylase in the planarian Dugesia japonica (DjTH) was identified. DjTH protein was coexpressed with aromatic amino acid decarboxylase-like A (DjAADCA) in the planarian central nervous system (CNS). In addition, DjTH-knockdown planarians lost the ability to synthesize DA, but showed no change in 5-hydroxytryptamine synthesis. When the planarian body was amputated, DjTH-positive neurons were regenerated in the brain newly rebuilt from the tail piece at Day 3, and the DjTH-positive axonal and dendritic neural network in the CNS (dopaminergic tiara) was reconstructed at Days 5-7. At that time, autonomic locomotion and methamphetamine-induced hyperkinesia were also suppressed in DjTH-knockdown planarians. Planarian locomotion and behavior seem to be regulated in both cilia- and muscle-dependent manners. In DjTH-knockdown planarians, muscle-mediated locomotion and behavior were significantly attenuated. These results suggest that DA neurons play a key role in the muscle-mediated movement in planarians.     

Zebrafish Parla- and Parlb-deficiency affects dopaminergic neuron patterning and embryonic survival

Many genes associated with familial Parkinson's disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson's disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson's disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.     

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.     

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.     

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Recent studies suggest that l-3,4 dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a severe complication of conventional L-DOPA therapy of Parkinson's disease, may be caused by dopamine (DA) release originating in serotonergic neurons. To evaluate the in vivo effect of a 5-HT(1A) agonist [(±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide, 8-OHDPAT] on the L-DOPA-induced increase in extracellular DA and decrease in [(11) C]raclopride binding in an animal model of advanced Parkinson's disease and LID, we measured extracellular DA in response to L-DOPA or a combination of L-DOPA and the 5-HT(1A) agonist, 8-OHDPAT, with microdialysis, and determined [(11) C]raclopride binding to DA receptors, with micro-positron emission tomography, as the surrogate marker of DA release. Rats with unilateral 6-hydroxydopamine lesions had micro-positron emission tomography scans with [(11) C]raclopride at baseline and after two pharmacological challenges with L-DOPA?+?benserazide with or without 8-OHDPAT co-treatment. Identical challenge regimens were used with the subsequent microdialysis concomitant with ratings of LID severity. The baseline increase of [(11) C]raclopride-binding potential (BP(ND) ) in lesioned striatum was eliminated by the L-DOPA challenge, while the concurrent administration of 8-OHDPAT prevented this L-DOPA-induced displacement of [(11) C]raclopride significantly in lesioned ventral striatum and near significantly in the dorsal striatum. With microdialysis, the L-DOPA challenge raised the extracellular DA in parallel with the emergence of strong LID. Co-treatment with 8-OHDPAT significantly attenuated the release of extracellular DA and LID. The 8-OHDPAT co-treatment reversed the L-DOPA-induced decrease of [(11) C]raclopride binding and increase of extracellular DA and reduced the severity of LID. The reversal of the effect of L-DOPA on [(11) C]raclopride binding, extracellular DA and LID by 5-HT agonist administration is consistent with the notion that part of the DA increase associated with LID originates in serotonergic neurons.     

Immortalization of neuronal progenitors using SV40 large T antigen and differentiation towards dopaminergic neurons

Transplantation is common in clinical practice where there is availability of the tissue and organ. In the case of neurodegenerative disease such as Parkinson's disease (PD), transplantation is not possible as a result of the non‐availability of tissue or organ and therefore, cell therapy is an innovation in clinical practice. However, the availability of neuronal cells for transplantation is very limited. Alternatively, immortalized neuronal progenitors could be used in treating PD. The neuronal progenitor cells can be differentiated into dopaminergic phenotype. Here in this article, the current understanding of the molecular mechanisms involved in the differentiation of dopaminergic phenotype from the neuronal progenitors immortalized with SV40 LT antigen is discussed. In addition, the methods of generating dopaminergic neurons from progenitor cells and the factors that govern their differentiation are elaborated. Recent advances in cell‐therapy based transplantation in PD patients and future prospects are discussed.     

X‐ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons

Transition metals have been suggested to play a pivotal role in the pathogenesis of Parkinson's disease. X‐ray microscopy combined with a cryogenic setup is a powerful method for elemental imaging in low concentrations and high resolution in intact cells, eliminating the need for fixation and sectioning of the specimen. Here, we performed an elemental distribution analysis in cultured primary midbrain neurons with a step size in the order of 300 nm and ~ 0.1 ppm sensitivity under cryo conditions by using X‐ray fluorescence microscopy. We report the elemental mappings on the subcellular level in primary mouse dopaminergic (DAergic) and non‐DAergic neurons after treatment with transition metals. Application of Fe²⁺ resulted in largely extracellular accumulation of iron without preference for the neuronal transmitter subtype. A quantification of different Fe oxidation states was performed using X‐ray absorption near edge structure analysis. After treatment with Mn²⁺, a cytoplasmic/paranuclear localization of Mn was observed preferentially in DAergic neurons, while no prominent signal was detectable after Mn³⁺ treatment. Immunocytochemical analysis correlated the preferential Mn uptake to increased expression of voltage‐gated calcium channels in DAergic neurons. We discuss the implications of this differential elemental distribution for the selective vulnerability of DAergic neurons and Parkinson's disease pathogenesis.     

Voltage-dependent and -independent block of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor channels

Polyamine-containing toxins and synthetic dicationic derivatives of adamantane and phenylcyclohexyl selectively antagonize Ca(2+)-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor channels. These compounds demonstrate voltage-dependent open-channel block and are trapped by closed channels. In this study, we describe an alternative mechanism of non-competitive AMPA receptor inhibition caused by 9-aminoacridine and some of its derivatives. These compounds exhibit similar potency against Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors. The inhibition is largely voltage-independent, binding and unbinding do not require presence of agonist. We conclude that 9-aminoacridine binds to a shallow site in the AMPA receptor, which is located above the activation gate. A comparison of three-dimensional structures of the antagonists suggests that the 'V-like' shape of the hydrophobic headgroup favors voltage-dependent binding to the deep site in the channel pore, whereas the compounds possessing flat aromatic headgroups preferably bind to the shallow site. The characterization of the novel mechanism of AMPA receptor channel antagonism opens a way to develop a new family of pharmacological agents, which can be of scientific and practical importance.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra,but are not required for axon degeneration

Activation of c‐jun N‐terminal kinase (JNK) by the mitogen‐activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson’s disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6‐hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.     

Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6-hydroxydopamine

Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6-hydroxydopamine (6-OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6-OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn-superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad-hSOD1). Survival of the dopaminergic cells exposed to 6-OHDA was 50% higher among the SOD1-producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6-OHDA)-treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H(2) O(2) -scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6-OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.     

Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

(G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD

(G2019S) mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurons and parkinsonism phenotypes of motor dysfunction. LRRK2 is a member of mixed lineage kinase subfamily of mitogen-activated protein kinase kinase kinases (MAPKKKs). We hypothesized that (G2019S) mutation augmented LRRK2 kinase activity, leading to overphosphorylation of downstream MAPK kinase (MKK) and resulting in activation of neuronal death signal pathway. Consistent with our hypothesis, (G2019S) LRRK2 expressed in HEK 293 cells exhibited an augmented kinase activity of phosphorylating MAPK kinase 4 (MKK4) at Ser(257), and protein expression of active phospho-MKK4(Ser257) was upregulated in the SN of (G2019S) LRRK2 transgenic mice. Protein level of active phospho-JNK(Thr183/Tyr185) and phospho-c-Jun(Ser63), downstream targets of phospho-MKK4(Ser257), was increased in the SN of (G2019S) LRRK2 mice. Upregulated mRNA expression of pro-apoptotic Bim and FasL, target genes of phospho-c-Jun(Ser63), and formation of active caspase-9, caspase-8 and caspase-3 were also observed in the SN of (G2019S) LRRK2 transgenic mice. Our results suggest that mutant (G2019S) LRRK2 activates MKK4-JNK-c-Jun pathway in the SN and causes the resulting degeneration of SNpc dopaminergic neurons in PD transgenic mice.     

Protective effect of orexin-A on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by progressive and selective death of midbrain dopaminergic neurons. Pharmacologic treatment of PD can be divided into symptomatic and neuroprotective therapies.