Role of nitric oxide in a progressive neurodegeneration model of Parkinson's disease in the rat

Abstract

This study was undertaken to investigate the nitric oxide synthase (NOS) activity in the striatum following 6-hydroxydopamine (6-OHDA) induced neurodegeneration in rats. Constitutive NOS (cNOS) activity remained unaltered at 3, 7 and 14 days after lesion, while a 43% and 45% decrease was observed at 30 and 50 days, respectively. Inducible NOS (iNOS) activity was detected only on the 3rd day after lesion and not in subsequent days or the control striatum. N^G-nitro-L-arginine methyl ester (L-NAME) pretreatment blocked the amphetamine-induced rotations and inhibited the iNOS activity at the 3rd day after the 6-OHDA injection. L-NAME pretreatment also significantly restored the striatal dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in 6-OHDA treated rats. Thus a possible role of nitric oxide in 6-OHDA induced neurodegeneration is suggested.     

Edaravone guards dopamine neurons in a rotenone model for Parkinson's disease

3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), an effective free radical scavenger, provides neuroprotection in stroke models and patients. In this study, we investigated its neuroprotective effects in a chronic rotenone rat model for Parkinson's disease. Here we showed that a five-week treatment with edaravone abolished rotenone's activity to induce catalepsy, damage mitochondria and degenerate dopamine neurons in the midbrain of rotenone-treated rats. This abolishment was attributable at least partly to edaravone's inhibition of rotenone-induced reactive oxygen species production or apoptotic promoter Bax expression and its up-regulation of the vesicular monoamine transporter 2 (VMAT2) expression. Collectively, edaravone may provide novel clinical therapeutics for PD.     

Mesenchymal stem cell secretome protects against alpha-synuclein-induced neurodegeneration in a Caenorhabditis elegans model of Parkinson's disease

Background aimsThe capacity of the secretome from bone marrow-derived mesenchymal stem cells (BMSCs) to prevent dopaminergic neuron degeneration caused by overexpression of alpha-synuclein (α-syn) was explored using two Caenorhabditis elegans models of Parkinson's disease (PD).MethodsFirst, a more predictive model of PD that overexpresses α-syn in dopamine neurons was subjected to chronic treatment with secretome. This strain displays progressive dopaminergic neurodegeneration that is age-dependent. Following chronic treatment with secretome, the number of intact dopaminergic neurons was determined. Following these initial experiments, a C. elegans strain that overexpresses α-syn in body wall muscle cells was used to determine the impact of hBMSC secretome on α-syn inclusions. Lastly, in silico analysis of the components that constitute the secretome was performed.ResultsThe human BMSC (hBMSC) secretome induced a neuroprotective effect, leading to reduced dopaminergic neurodegeneration. Moreover, in animals submitted to chronic treatment with secretome, the number of α-syn inclusions was reduced, indicating that the secretome of MSCs was possibly contributing to the degradation of those structures. In silico analysis identified possible suppressors of α-syn proteotoxicity, including growth factors and players in the neuronal protein quality control mechanisms.ConclusionsThe present findings indicate that hBMSC secretome has the potential to be used as a disease-modifying strategy in future PD regenerative medicine approaches.     

Imaging neurodegeneration in Parkinson's disease

Neuroimaging techniques have evolved over the past several years giving us unprecedented information about the degenerative process in Parkinson's disease (PD) and other movement disorders. Functional imaging approaches such as positron emission tomography (PET) and single photon emission computerised tomography (SPECT) have been successfully employed to detect dopaminergic dysfunction in PD, even while at a preclinical stage, and to demonstrate the effects of therapies on function of intact dopaminergic neurons within the affected striatum. PET and SPECT can also monitor PD progression as reflected by changes in brain levodopa and glucose metabolism and dopamine transporter binding. Structural imaging approaches include magnetic resonance imaging (MRI) and transcranial sonography (TCS). Recent advances in voxel-based morphometry and diffusion-weighted MRI have provided exciting potential applications for the differential diagnosis of parkinsonian syndromes. Substantia nigra hyperechogenicity, detected with TCS, may provide a marker of susceptibility to PD, probably reflecting disturbances of iron metabolism, but does not appear to correlate well with disease severity or change with disease progression. In the future novel radiotracers may help us assess the involvement of non-dopaminergic brain pathways in the pathology of both motor and non-motor complications in PD.     

Zonisamide reduces nigrostriatal dopaminergic neurodegeneration in a mouse genetic model of Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of nigrostriatal dopaminergic neurons and consequent motor dysfunction. Zonisamide (1,2‐benzisoxazole‐3‐methanesulfonamide), which was originally developed as an antiepileptic drug, has been found to have therapeutic benefits for PD. However, the pharmacological mechanisms behind the beneficial actions of zonisamide in PD are not fully understood. Here, we investigated the neuroprotective effects of zonisamide on nigrostriatal dopaminergic neurons of the Engrailed mutant mouse, a genetic model of PD. Chronic administration of zonisamide in Engrailed mutant mice was shown to improve the survival of nigrostriatal dopaminergic neurons compared with that under saline treatment. In addition, dopaminergic terminals in the striatum and the motor function were improved in zonisamide‐treated Engrailed mutant mice to the levels of those in control mice. To clarify the mechanism behind the neuroprotective effects of zonisamide, the contents of neurotrophic factors were determined after chronic administration of zonisamide. Brain‐derived neurotrophic factor content was increased in the striatum and ventral midbrain of the zonisamide‐treated mice compared to saline‐treated mice. These findings imply that zonisamide reduces nigrostriatal dopaminergic cell death through brain‐derived neurotrophic factor signaling and may have similar beneficial effects in human parkinsonian patients as well.

     

Ephrin-A1-mediated dopaminergic neurogenesis and angiogenesis in a rat model of Parkinson's disease

Cells of the neural stem cell lineage in the adult subventricular zone (SVZ) respond to brain insult by increasing their numbers and migrating through the rostral migratory stream. However, in most areas of the brain other than the SVZ and the subgranular zone of the dentate gyrus, such a regenerative response is extremely weak. Even these two neurogenic regions do not show extensive regenerative responses to repair tissue damage, suggesting the presence of an intrinsic inhibitory microenvironment (niche) for stem cells. In the present study, we assessed the effects of injection of clustered ephrin-A1-Fc into the lateral ventricle of rats with unilateral nigrostriatal dopamine depletion. Ephrin-A1-Fc clustered by anti-IgG(Fc) antibody was injected stereotaxically into the ipsilateral lateral ventricle of rats with unilateral nigrostriatal lesions induced by 6-hydroxydopamine, and histologic analysis and behavioral tests were performed. Clustered ephrin-A1-Fc transformed the subventricular niche, increasing bromodeoxyuridine-positive cells in the subventricular area, and the cells then migrated to the striatum and differentiated to dopaminergic neurons and astrocytes. In addition, clustered ephrin-A1-Fc enhanced angiogenesis in the striatum on the injected side. Along with histologic improvements, behavioral derangement improved dramatically. These findings indicate that the subventricular niche possesses a mechanism for regulating both stem cell and angiogenic responses via an EphA-mediated signal. We conclude that activation of EphA receptor-mediated signaling by clustered ephrin-A1-Fc from within the lateral ventricle could potentially be utilized in the treatment of neurodegenerative diseases such as Parkinson's disease.     

Macrophage antigen complex-1 mediates reactive microgliosis and progressive dopaminergic neurodegeneration in the MPTP model of Parkinson's disease

Neuronal death is known to trigger reactive microgliosis. However, little is known regarding the manner by which microglia are activated by injured neurons and how microgliosis participates in neurodegeneration. In this study we delineate the critical role of macrophage Ag complex-1 (MAC1), a member of the beta(2) integrin family, in mediating reactive microgliosis and promoting dopaminergic (DAergic) neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. MAC1 deficiency greatly attenuated the DAergic neurodegeneration induced by MPTP or 1-methyl-4-phenyl-pyridium iodide (MPP(+)) exposure both in vivo and in vitro, respectively. Reconstituted experiments created by adding microglia from MAC1(-/-) or MAC1(+/+) mice back to MAC1(+/+) neuron-enriched cultures showed that microglia with functional MAC1 expression was mandatory for microglia-enhanced neurotoxicity. Both in vivo and in vitro morphological and Western blot studies demonstrated that MPTP/MPP(+) produced less microglia activation in MAC1(-/-) mice than MAC1(+/+) mice. Further mechanistic studies revealed that a MPP(+)-mediated increase in superoxide production was reduced in MAC1(-/-) neuron-glia cultures compared with MAC1(+/+) cultures. The stunted production of superoxide in MAC1(-/-) microglia is likely linked to the lack of translocation of the cytosolic NADPH oxidase (PHOX) subunit (p47(phox)) to the membrane. In addition, the production of PGE(2) markedly decreased in neuron plus MAC1(-/-) microglia cocultures vs neuron plus MAC1(+/+) microglia cocultures. Taken together, these results demonstrate that MAC1 plays a critical role in MPTP/MPP(+)-induced reactive microgliosis and further support the hypothesis that reactive microgliosis is an essential step in the self-perpetuating cycle leading to progressive DAergic neurodegeneration observed in Parkinson's disease.     

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson''s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.     

PEP-1-PEA-15 protects against toxin-induced neuronal damage in a mouse model of Parkinson's disease

Background

PEA-15 is abundantly expressed in both neurons and astrocytes throughout the brain. It is a multifunctional protein with the ability to increase cell survival via anti-apoptotic and anti-proliferative properties. However, the function of PEA-15 in neuronal diseases such as Parkinson's disease (PD) remains unclear. In this study, we investigated the protective effects of PEA-15 on neuronal damage induced by MPP⁺ in neuroblastoma SH-SY5Y and BV2 microglia cells and in a MPTP-induced PD mouse model using cell-permeable PEP-1-PEA-15.

Methods

PEP-1-PEA-15 was purified using affinity chromatography. Cell viability and DNA fragmentation were examined by MTT assay and TUNEL staining. Dopaminergic neuronal cell death in the animal model was examined by immunohistochemistry.

Results

PEP-1-PEA-15 transduced into the SH-SY5Y and BV2 cells in a time- and dose-dependent manner. Transduced PEP-1-PEA-15 protected against MPP⁺-induced toxicity by inhibiting intracellular ROS levels and DNA fragmentation. Further, it enhanced the expression levels of Bcl-2 and caspase-3 while reducing the expression levels of Bax and cleaved caspase-3. We found that PEP-1-PEA-15 transduced into the substantia nigra and prevented dopaminergic neuronal cell death in a MPTP-induced PD mouse. Also, we showed the neuroprotective effects in the model by demonstrating that treatment with PEP-1-PEA-15 ameliorated MPTP-induced behavioral dysfunctions and increased dopamine levels in the striatum.

Conclusions

PEP-1-PEA-15 can efficiently transduce into cells and protects against neurotoxin-induced neuronal cell death in vitro and in vivo.

General significance

These results demonstrate the potential for PEP-1-PEA-15 to provide a new strategy for protein therapy treatment of a variety of neurodegenerative diseases including PD.     

Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson's disease produced by decreasing doses of alpha-synuclein

The pathological hallmarks of Parkinson's disease (PD) are degeneration of dopamine (DA) neurons of the substantia nigra (SN) and the presence of alpha-synuclein (α-syn)-rich Lewy bodies in DA cells that remain. To model these aspects of the disease, we previously showed that high titer (5.1×10exp12 gp/ml) AAV1/2 driven expression of A53T α-syn in the SN of rats caused nigrostriatal pathology including a loss of DA neurons, but also with toxicity in the GFP control group. In the current study, we evaluate the effects of two lower titers by dilution of the vector (1∶3 [1.7×10exp12] and 1∶10 [5.1×10exp11]) to define a concentration that produced pathology specific for α-syn. In GFP and empty vector groups there were no behavioural or post-mortem changes at 3 or 6 weeks post-administration at either vector dose. Dilution of the AAV1/2 A53T α-syn (1:3) produced significant paw use asymmetry, reductions in striatal tyrosine hydroxylase (TH), and increases in DA turnover at 3 weeks in the absence of overt pathology. By 6 weeks greater evidence of pathology was observed and included, reductions in SN DA neurons, striatal DA, TH and DA-transporter, along with a sustained behavioural deficit. In contrast, the 1:10 AAV1/2 A53T α-syn treated animals showed normalization between 3 and 6 weeks in paw use asymmetry, reductions in striatal TH, and increased DA turnover. Progression of dopaminergic deficits using the 1:3 titer of AAV1/2 A53Tα-syn provides a platform for evaluating treatments directed at preventing and/or reversing synucleinopathy. Use of the 1:10 titer of AAV1/2 A53T α-syn provides an opportunity to study mechanisms of endogenous compensation. Furthermore, these data highlight the need to characterize the titer of vector being utilized, when using AAV to express pathogenic proteins and model disease process, to avoid producing non-specific effects.     

Synergistic effects of environmental risk factors and gene mutations in Parkinson's disease accelerate age-related neurodegeneration

As Parkinson's disease appears to be a multifactoral disorder, the use of animal models to investigate combined effects of genetic and environmental risk factors are of great importance especially in the context of aging which is the single major risk factor for the disorder. Here, we assessed the combined effects of neonatal iron feeding and environmental paraquat exposure on age-related nigrostriatal degeneration in transgenic mice expressing the A53T familial mutant form of human α-synuclein within these neurons. We report here that A53T α-synuclein mice exhibit greater susceptibility to paraquat. Increased oral intake of iron in the neonatal period leads to a progressive age-related enhancement of dopaminergic neurodegeneration associated with paraquat neurotoxicity. Furthermore, neurodegeneration associated with these combined genetic and environmental risk factors could be attenuated by systemic treatment with the bioavailable antioxidant compound EUK-189. These data suggest that environmental factors previously identified as contributors to neurodegeneration associated with sporadic Parkinson's disease may also be candidates for observed variations in symptoms and disease progression in monogenic forms and that this may mechanistically involve increased levels of oxidatively-induced post-translational nitration of α-synuclein.     

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.     

PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.     

SIRT1 attenuates neuroinflammation by deacetylating HSPA4 in a mouse model of Parkinson's disease

As a deacetylase, SIRT1 plays essential roles in various physiological events, from development to lifespan regulation. SIRT1 has been shown neuroprotective effects in neurodegeneration disorders such as Parkinson's disease (PD). However, the underlying molecular mechanisms are still not well understood. Here, we generated transgenic mice with increased expression of Sirt1 in the brain and examined the potential roles of SIRT1 in PD. Our data showed that SIRT1 repressed proinflammatory cytokine expression both in microglia and astrocytes. In MPTP induced PD model mice, lower levels of microglia and astrocyte activation were observed in SIRT1 transgenic mice. Moreover, the tyrosine hydroxylase (TH) loss in the substantia nigra pars compacta (SNpc) and striatum induced by MPTP was also attenuated by SIRT1. As a consequence, the behavioral defects induced by MPTP were largely prevented in SIRT1 transgenic mice. Mechanistically, SIRT1 interacts with heat shock 70 kDa protein 4 (HSPA4) and deacetylates it at 305, 351 and 605 lysine residues. This deacetylation modification induces the nuclear translocation of HSPA4 and thus to repress proinflammatory cytokine expression. On the contrary, mutated HSPA4, in which 305/351/605 lysine residues were replaced with arginine, was mainly localized in the cytoplasm and losses its repression on proinflammatory cytokine expression. Taken together, our data indicate that SIRT1 plays beneficial roles in PD model mice, which is likely due to, at least in part, its anti-inflammation activity in glial cells by deacetylating HSPA4. Furthermore, HSPA4 might be a druggable target for developing novel agents for treating neuroinflammation associated disorders such as PD.