Angiogenin in Parkinson Disease Models: Role of Akt Phosphorylation and Evaluation of AAV-Mediated Angiogenin Expression in MPTP Treated Mice

The angiogenic factor, angiogenin, has been recently linked to both Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). We have recently shown that endogenous angiogenin levels are dramatically reduced in an alpha-synuclein mouse model of PD and that exogenous angiogenin protects against cell loss in neurotoxin-based cellular models of PD. Here, we extend our studies to examine whether activation of the prosurvival Akt pathway is required for angiogenin''s neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+), as observed in ALS models, and to test the effect of virally-mediated overexpression of angiogenin in an in vivo PD model. Using a dominant negative Akt construct, we demonstrate that inhibition of the Akt pathway does not reduce the protective effect of angiogenin against MPP+ toxicity in the dopaminergic SH-SY5Y cell line. Furthermore, an ALS-associated mutant of angiogenin, K40I, which fails to induce Akt phosphorylation, was similar to wildtype angiogenin in protection against MPP+. These results confirm previous work showing neuroprotective effects of angiogenin against MPP+, and indicate that Akt is not required for this protective effect. We also investigated whether adeno-associated viral serotype 2 (AAV2)-mediated overexpression of angiogenin protects against dopaminergic neuron loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. We found that angiogenin overexpression using this approach does not reduce the MPTP-induced degeneration of dopaminergic cells in the substantia nigra, nor limit the depletion of dopamine and its metabolites in the striatum. Together, these findings extend the evidence for protective effects of angiogenin in vitro, but also suggest that further study of in vivo models is required to translate these effects into meaningful therapies.     

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.     

Reactive Macrophages Increase Oxidative Stress and Alpha-Synuclein Nitration During Death of Dopaminergic Neuronal Cells in Co-Culture: Relevance to Parkinson’s Disease

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1400W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.     

Inhibition of fibrillization and accumulation of prefibrillar oligomers in mixtures of human and mouse alpha-synuclein

Parkinson's disease (PD) is a neurodegenerative disorder attributed to the loss of dopaminergic neurons from the substantia nigra. Some surviving neurons are characterized by cytoplasmic Lewy bodies, which contain fibrillar alpha-synuclein. Two mutants of human alpha-synuclein (A53T and A30P) have been linked to early-onset, familial PD. Oligomeric forms of these mutants accumulate more rapidly and/or persist for longer periods of time than oligomeric, human wild-type alpha-synuclein (WT), suggesting a link between oligomerization and cell death. The amino acid sequences of the mouse protein and WT differ at seven positions. Mouse alpha-synuclein, like A53T, contains a threonine residue at position 53. We have assessed the conformational properties and fibrillogenicity of the murine protein. Like WT and the two PD mutants, mouse alpha-synuclein adopts a "natively unfolded" or disordered structure. However, at elevated concentrations, the mouse protein forms amyloid fibrils more rapidly than WT, A53T, or A30P. The fibrillization of mouse alpha-synuclein is slowed by WT and A53T. Inhibition of fibrillization leads to the accumulation of nonfibrillar, potentially toxic oligomers. The results are relevant to the interpretation of the phenotypes of transgenic animal models of PD and suggest a novel approach for testing the cause and effect relationship between fibrillization and neurodegeneration.     

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.     

Transduced Tat-DJ-1 protein protects against oxidative stress-induced SH-SY5Y cell death and Parkinson disease in a mouse model

Parkinson's disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD.     

rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra.

Results

We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants.

Conclusions

In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7.

     

Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson's disease.

Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.     

Effect of the overexpression of wild-type or mutant alpha-synuclein on cell susceptibility to insult

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.     

Deletion of alpha-synuclein decreases impulsivity in mice

The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication.     

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4-phenylbutyrate (4-PBA) on nigral dopamine (DA) neurons in rotenone-treated mice. 4-PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up-regulation of alpha-synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4-PBA inhibited rotenone-induced neuronal death and decreased the protein level of alpha-synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4-PBA has a neuroprotective effect in the treatment of PD.     

Dyrk1A phosphorylates alpha-synuclein and enhances intracellular inclusion formation

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.     

alpha-synuclein and Parkinson's disease: the first roadblock

alpha-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and alpha-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of alpha-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed alpha- synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. alpha-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that alpha- synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of alpha-synuclein-induced neurodegeneration. alpha-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of alpha-synuclein with components of neuronal membrane traffic.     

Expression of alpha-synuclein in non-apoptotic,slowly degenerating facial motoneurones

The discovery that missense mutations in the alpha-synuclein gene represent a rare genetic cause of Parkinson's disease (PD) has had significant impact on the development of research into neurodegenerative disorders. It is becoming increasingly clear that alpha-synuclein plays a central role in the pathological process, which causes Lewy body formation and neurodegeneration in PD. Importantly, there is evidence to suggest that mutated alpha-synuclein is toxic to both nerve cells and glia. However, the regulation and function of wild-type alpha-synuclein are as yet ill defined. Using the facial nerve axotomy model, we have addressed the question whether the expression of alpha-synuclein in nerve cells may change in response to injury. We were particularly interested in testing the hypothesis that the severity of neuronal injury had an effect on alpha-synuclein metabolism. Facial nerve cut and crush, respectively, were performed in adult rats where normal facial motoneurones do not express alpha-synuclein. Following axotomy, a subset of facial motoneurones newly expressed high levels of alpha-synuclein immunoreactivity in their cell body and, occasionally, their nucleus. Significantly more nerve cells were labelled following facial nerve transection than following facial nerve crush. Confocal microscopy revealed a granular pattern of alpha-synuclein aggregation in degenerating nerve cells. Interestingly, the observed cell death phenotype was clearly non-apoptotic and developed over days or weeks rather than hours. Thus, axotomy of adult rat facial motoneurones triggers de novo expression of alpha-synuclein and this expression is associated with a non-apoptotic, slow form a neurodegeneration. In addition, the extent of alpha-synuclein expression is related to the severity of neuronal injury.     

Angiotensin II protects against alpha-synuclein toxicity and reduces protein aggregation in vitro

In this study, we examined the effects of angiotensin II (AngII) in a genetic in vitro PD model produced by alpha-synuclein (alpha-syn) overexpression in the human neuroglioma H4 cell line. We observed a maximal decrease in alpha-syn-induced toxicity of 85% and reduction in inclusion formation by 19% when cultures were treated with AngII in the presence of the angiotensin type 1 (AT1) receptor antagonist losartan and AT2 receptor antagonist PD123319. When compared to AngII, the AT4 receptor agonist AngIV was moderately effective in protecting H4 cells against alpha-syn toxicity and did not significantly reduce inclusion formation. Here we show that AngII is protective against genetic, as well as neurotoxic models of PD. These data support the view that agents acting on the renin-angiotensin-system (RAS) may be useful in the prevention and/or treatment of Parkinson's disease.     

Impairment of redox state and dopamine level induced by alpha-synuclein aggregation and the prevention effect of hsp70

One hypothesis for the etiology of Parkinson's disease (PD) is that the formation of proteinaceous inclusion, which is mainly composed of alpha-synuclein, may contribute to the selective loss of dopaminergic neurons. To further explore the role of alpha-synuclein in neurodegeneration of PD, we examined the possible effects of aggregated alpha-synuclein on the intracellular redox state, dopamine level, and cell death of SK-N-SH cells. Our present studies show that alpha-synuclein aggregation gives rise to both elevated intracellular oxidative state and dopamine level in SK-N-SH cells. Moreover, alpha-synuclein aggregation results in a higher ratio of apoptosis population (55.8%+/-SEM) in cells overexpressing alpha-synuclein aggregation, compared to their normal control groups (8.0%+/-SEM). In contrast, coexpression of hsp70 with alpha-synuclein suppresses the oxidative state shift, restores the normal dopamine levels and blocks neuron cell loss. Therefore, our data provided one possible mechanism by which the alpha-synuclein aggregation may lead to the neurodegeneration in PD via regulating the level of cytoplasmic dopamine and then disturbing the intracellular redox homeostasis. On the other hand, hsp70 can mitigate the degenerative effect conferred by alpha-synuclein, acting as a protective factor in treatment of PD.     

Application of proteasomal inhibitors to mouse sympathetic neurons activates the intrinsic apoptotic pathway

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibition-induced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria.