Pathogenic Mutation in VPS35 Impairs Its Protection against MPP+ Cytotoxicity

Parkinson''s disease primarily results from progressive degeneration of dopaminergic neurons in the substantia nigra. Both neuronal toxicants and genetic factors are suggested to be involved in the disease pathogenesis. The mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP⁺) shows a highly selective toxicity to dopaminergic neurons. Recent studies indicate that mutation in the vacuolar protein sorting 35 (vps35) gene segregates with Parkinson''s disease in some families, but how mutation in the vps35 gene causes dopaminergic cell death is not known. Here, we report that enhanced VPS35 expression protected dopaminergic cells against MPP⁺ toxicity and that this neuroprotection was compromised by pathogenic mutation in the gene. A loss of neuroprotective functions contributes to the pathogenesis of VPS35 mutation in Parkinson''s disease.     

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1‐induced precursors expressed dopaminergic markers and exhibited depolarization‐evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1‐induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine‐induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.     

Molecular Docking-Based Identification of Potential Natural Neuroprotective Molecules for Parkinson's Disease

Background: Parkinson's disease (PD) is a common progressive neurodegenerative and the prevailing treatments are ineffective in the early stages of the disease. Therefore, other strategies must be devised to halt the steady decrease of dopaminergic neurons in the brain. In Parkinson's disease, a dysregulated ACE/Ang II/AT1R axis in the brain causes free radical damage, apoptosis, and neuronal destruction. Current PD treatments only alleviate symptoms and do not reverse the degradation mechanism of dopaminergic neurons. As a result, it is critical to discover alternate, dependable medicines for the treatment of Parkinson's disease. Method : In the present study, homology modelling of MAS receptor, in silico docking and molecular dynamic studies (MDS) were employed to determine the efficacy of flavonoids as MASR activators. Result : The flavonoids Pterosupin and Amentoflavone exhibited best binding and therefore, the stability of these complexes were evaluated with MDS studies. The Pterosupin-MASR complex demonstrated better stability, stronger interactions and minimal fluctuation than the Amentoflavone-MASR complex. Conclusion : The data from the present study indicated that the flavonoid Pterosupin possesses better binding, favourable pharmacokinetic properties and stability. However, subsequent in vitro and in vivo assessments are necessary to validate its efficacy.     

Preventive role of PD‐1 on MPTP‐induced dopamine depletion in mice

Many current studies of Parkinson's disease (PD) suggest that inflammation is involved in the neurodegenerative process. PD‐1, a traditional Korean medicine, used to treat various brain diseases in Korea. This study was designed to investigate the effect of PD‐1 extract in the Parkinson's model of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) lesioned mice. The MPTP administration caused the dopamine neuron loss in the striatum and substantia nigra pars compacta (SNpc), which was demonstrated by a depletion of tyrosine hydroxylase (TH). In addition, a reduction of bcl‐2 expression with elevation of bax expression, caspase‐3 activation, and release of cytochrome c into cytosol in dopaminergic neurons of SNpc were noted. Oral administration of PD‐1 extract (50 and 100 mg kg^?1) attenuated the MPTP‐induced depletion of TH proteins in the striatum and SNpc and prevented the apoptotic effects. These results indicate that PD‐1 extract is able to protect dopaminergic neurons from MPTP‐induced neuronal death, with important implications for the treatment of PD. Copyright © 2010 John Wiley & Sons, Ltd.     

Increased isoprostane and prostaglandin are prominent in neurons in Alzheimer disease

Parkinson's disease is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. Loss-of-function mutations in the DJ-1 gene have been linked to autosomal recessive forms of early-onset familial Parkinson's disease. DJ-1 is thought to play roles in protection of cells against oxidative stress and in maintenance of the normal dopaminergic function in the nigrostriatal pathway. Here we investigate the consequence of both DJ-1 inactivation and aging in mice. We found that DJ-1-/- mice at the age of 24–27 months have normal numbers of dopaminergic neurons in the substantia nigra and normal levels of dopamine and its major metabolites in the striatum. The number of noradrenergic neurons in the locus coeruleus is also unchanged in DJ-1-/- mice. Moreover, there is no accumulation of oxidative damage or inclusion bodies in aged DJ-1-/- brains. Together, these results indicate that loss of DJ-1 function alone is insufficient to cause nigral degeneration and oxidative damage in the life span of mice.     

Increased Ndfip1 in the Substantia Nigra of Parkinsonian Brains Is Associated with Elevated Iron Levels

Iron misregulation is a central component in the neuropathology of Parkinson''s disease. The iron transport protein DMT1 is known to be increased in Parkinson''s brains linking functional transport mechanisms with iron accumulation. The regulation of DMT1 is therefore critical to the management of iron uptake in the disease setting. We previously identified post-translational control of DMT1 levels through a ubiquitin-mediated pathway led by Ndfip1, an adaptor for Nedd4 family of E3 ligases. Here we show that loss of Ndfip1 from mouse dopaminergic neurons resulted in misregulation of DMT1 levels and increased susceptibility to iron induced death. We report that in human Parkinson''s brains increased iron concentrations in the substantia nigra are associated with upregulated levels of Ndfip1 in dopaminergic neurons containing α-synuclein deposits. Additionally, Ndfip1 was also found to be misexpressed in astrocytes, a cell type normally devoid of this protein. We suggest that in Parkinson''s disease, increased iron levels are associated with increased Ndfip1 expression for the regulation of DMT1, including abnormal Ndfip1 activation in non-neuronal cell types such as astrocytes.     

Parkin-Dependent Degradation of the F-Box Protein Fbw7β Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1

Parkinson''s disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson''s disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCF^Fbw7β. Specifically, parkin targets the SCF substrate adapter Fbw7β for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7β levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCF^Fbw7β target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCF^Fbw7β-mediated ubiquitylation-dependent proteolysis of Mcl-1.     

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron‐like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule‐associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β‐tubulin III). It also increased expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine‐beta‐hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.     

Regulation of c‐Ret,GFRα1, and GFRα2 in the substantia nigra Pars compacta in a rat model of Parkinson's disease

Glial cell line‐derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6‐hydroxydopamine (6‐OHDA) transiently increased c‐Ret and GFRα1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c‐Ret and GFRα1 was downregulated. GFRα2 expression was differentially regulated, as it decreased only 6 days after 6‐OHDA injection. Triple‐labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6‐OHDA lesion. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 343–351, 2002     

Irreversible Inhibition of Mitochondrial Complex I by 7-(2-Aminoethyl)-3,4-Dihydro-5-Hydroxy-2H-1,4-Benzothiazine-3-Carboxylic Acid (DHBT-1): A Putative Nigral Endotoxin of Relevance to Parkinson's Disease

Abstract: Based on a number of lines of evidence, we have proposed recently that a very early step in the pathogenesis of idiopathic Parkinson's disease might be elevated translocation of l -cysteine into neuromelanin-pigmented dopaminergic cell bodies in the substantia nigra. In vitro studies suggest that such an influx of l -cysteine would divert the neuromelanin pathway by scavenging dopamine-o-quinone, the proximate autoxidation product of dopamine, to give 5-S-cysteinyldopamine, which is oxidized further to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) and other cysteinyldopamines and dihydrobenzothiazines. In this study, it is demonstrated that DHBT-1 inhibits ADP-stimulated oxidation of malate and pyruvate (state 3 or complex I respiration) when incubated with intact rat brain mitochondria with an IC₅₀ of ～0.80 mM. Incubation of DHBT-1 with freeze-thawed rat brain mitochondria in both the presence and absence of KCN and/or NADH causes an irreversible, time-dependent decrease of NADH-coenzyme Q₁ reductase activity. Significantly lower concentrations of DHBT-1 are necessary to cause this effect when mitochondrial membranes are incubated in the absence of KCN and NADH. The irreversible inhibition of mitochondrial complex I caused by DHBT-1 under the latter conditions could be blocked only partially by glutathione, ascorbic acid, superoxide dismutase, or catalase. Together, these results suggest that DHBT-1 can cross the outer mitochondrial membrane and irreversibly inhibit complex I by a mechanism that is not primarily related to oxygen radical-mediated damage. Formation of DHBT-1 requires only dopamine, l -cysteine, and an oxidizing environment, conditions that may well exist in the cytoplasm of neuromelanin-pigmented dopaminergic neurons in the parkinsonian substantia nigra. The results of this study raise the possibility that DHBT-1 might be an endotoxin formed specifically in pigmented dopaminergic neurons that can contribute to irreversible damage to mitochondrial complex I and substantia nigra cell death in Parkinson's disease.     

Membrane phospholipid peroxidation promotes loss of dopaminergic neurons in psychological stress-induced Parkinson's disease susceptibility

Parkinson's disease (PD) is a neurodegenerative disorder associated with α-synuclein aggregation and dopaminergic neuron loss in the midbrain. There is evidence that psychological stress promotes PD progression by enhancing glucocorticoids-related oxidative damage, however, the mechanisms involved are unknown. The present study demonstrated that plasma membrane phospholipid peroxides, as determined by phospholipidomics, triggered ferroptosis in dopaminergic neurons, which in turn contributed to stress exacerbated PD-like motor disorder in mice overexpressing mutant human α-synuclein. Using hormonomics, we identified that stress stimulated corticosteroid release and promoted 15-lipoxygenase-1 (ALOX15)-mediated phospholipid peroxidation. ALOX15 was upregulated by α-synuclein overexpression and acted as a fundamental risk factor in the development of chronic stress-induced parkinsonism and neurodegeneration. Further, we demonstrated the mechanism by which corticosteroids activated the PKC pathway and induced phosphatidylethanolamine-binding protein-1 (PEBP1) to form a complex with ALOX15, thereby facilitating ALOX15 to locate on the plasma membrane phospholipids. A natural product isolated from herbs, leonurine, was screened with activities of inhibiting the ALOX15/PEBP1 interaction and thereby attenuating membrane phospholipid peroxidation. Collectively, our findings demonstrate that stress increases the susceptibility of PD by driving membrane lipid peroxidation of dopaminergic neurons and suggest the ALOX15/PEBP1 complex as a potential intervention target.     

Alpha-Synuclein Disrupted Dopamine Homeostasis Leads to Dopaminergic Neuron Degeneration in Caenorhabditis elegans

Disruption of dopamine homeostasis may lead to dopaminergic neuron degeneration, a proposed explanation for the specific vulnerability of dopaminergic neurons in Parkinson''s disease. While expression of human α-synuclein in C. elegans results in dopaminergic neuron degeneration, the effects of α-synuclein on dopamine homeostasis and its contribution to dopaminergic neuron degeneration in C. elegans have not been reported. Here, we examined the effects of α-synuclein overexpression on worm dopamine homeostasis. We found that α-synuclein expression results in upregulation of dopamine synthesis and content, and redistribution of dopaminergic synaptic vesicles, which significantly contribute to dopaminergic neuron degeneration. These results provide in vivo evidence supporting a critical role for dopamine homeostasis in supporting dopaminergic neuron integrity.     

H2O2- or l-DOPA-injured dopaminergic neurons trigger the release of soluble mediators that up-regulate striatal GDNF through different signalling pathways

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective molecule for dopaminergic neurons of the nigrostriatal pathway that degenerate in Parkinson's disease. We have previously shown that H₂O₂- or l-3,4-dihydroxyphenylalanine (l-DOPA)-challenged dopaminergic neurons trigger the release of soluble factors that signal ventral midbrain astrocytes to increase GDNF expression. In the present work, we evaluated whether the factors released by ventral midbrain-challenged cells were able to alter GDNF expression in striatal cells, the targets of dopaminergic neurons projecting from the substantia nigra, and investigated the signalling pathways involved. Our data showed that soluble mediators released upon H₂O₂- or l-DOPA-induced dopaminergic injury up-regulated GDNF in striatal cells, with different temporal patterns depending on the oxidative agent used. Conditioned media from H₂O₂- or l-DOPA-challenged midbrain astrocyte cultures failed to up-regulate GDNF in striatal cultures. Likewise, there was no direct effect of H₂O₂ or l-DOPA on striatal GDNF levels suggesting that GDNF up-regulation was mediated by soluble factors released in the presence of failing dopaminergic neurons. Both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways were involved in striatal GDNF up-regulation triggered by H₂O₂-induced dopaminergic injury, while diffusible factors released in the presence of l-DOPA-challenged dopaminergic neurons induced GDNF expression in striatal cells through the activation of the MAPK pathway. These soluble mediators may constitute, in the future, important targets for the control of endogenous GDNF expression enabling the development of new and, hopefully, more efficient neuroprotective/neurorestorative strategies for the treatment of Parkinson's disease.     

Protective and toxic roles of dopamine in Parkinson's disease

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ‐1 (PARK7), ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL‐1), serine/threonine‐protein kinase (PINK‐1), leucine‐rich repeat kinase 2 (LRRK2), cation‐transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o‐quinone, aminochrome and 5,6‐indolequinone are formed. However, both dopamine o‐quinone and 5,6‐indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o‐quinone, aminochrome and 5,6‐indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o‐quinone, aminochrome and 5,6‐indolequinone polymerization to neuromelanin and (iii) two‐electron reduction of aminochrome catalyzed by DT‐diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context.

     

Intranasal administration of α-synuclein preformed fibrils triggers microglial iron deposition in the substantia nigra of Macaca fascicularis

Iron deposition is present in main lesion areas in the brains of patients with Parkinson’s disease (PD) and an abnormal iron content may be associated with dopaminergic neuronal cytotoxicity and degeneration in the substantia nigra of the midbrain. However, the cause of iron deposition and its role in the pathological process of PD are unclear. In the present study, we investigated the effects of the nasal mucosal delivery of synthetic human α-synuclein (α-syn) preformed fibrils (PFFs) on the pathogenesis of PD in Macaca fascicularis. We detected that iron deposition was clearly increased in a time-dependent manner from 1 to 17 months in the substantia nigra and globus pallidus, highly contrasting to other brain regions after treatments with α-syn PFFs. At the cellular level, the iron deposits were specifically localized in microglia but not in dopaminergic neurons, nor in other types of glial cells in the substantia nigra, whereas the expression of transferrin (TF), TF receptor 1 (TFR1), TF receptor 2 (TFR2), and ferroportin (FPn) was increased in dopaminergic neurons. Furthermore, no clear dopaminergic neuron loss was observed in the substantia nigra, but with decreased immunoreactivity of tyrosine hydroxylase (TH) and appearance of axonal swelling in the putamen. The brain region-enriched and cell-type-dependent iron localizations indicate that the intranasal α-syn PFFs treatment-induced iron depositions in microglia in the substantia nigra may appear as an early cellular response that may initiate neuroinflammation in the dopaminergic system before cell death occurs. Our data suggest that the inhibition of iron deposition may be a potential approach for the early prevention and treatment of PD.Subject terms: Parkinson''s disease, Parkinson''s disease     

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background

The pathology of Parkinson''s disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinson''s disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown.

Methodology/Principal Findings

In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D₂R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects.

Conclusions/Significance

Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.