Primary Cultures Of Mesencephalic Neurons Research Articles

Fyn Kinase-Mediated PKCδ Y311 Phosphorylation Induces Dopaminergic Degeneration in Cell Culture and Animal Models: Implications for the Identification of a New Pharmacological Target for Parkinson's Disease.

Oxidative stress, neuroinflammation and apoptosis are some of the key etiological factors responsible for dopamin(DA)ergic degeneration during Parkinson’s disease (PD), yet the downstream molecular mechanisms underlying neurodegeneration are largely unknown. Recently, a genome-wide association study revealed the FYN gene to be associated with PD, suggesting that Fyn kinase could be a pharmacological target for PD. In this study, we report that Fyn-mediated PKCδ tyrosine (Y311) phosphorylation is a key event preceding its proteolytic activation in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinsonism. MPP+/MPTP induced Fyn kinase activation in N27 DAergic neuronal cells and the mouse substantia nigra. PKCδ-Y311 phosphorylation by activated Fyn initiates the apoptotic caspase-signaling cascade during DAergic degeneration. Pharmacological attenuation of Fyn activity protected DAergic neurons from MPP+-induced degeneration in primary mesencephalic neuronal cultures. We further employed Fyn wild-type and Fyn knockout (KO) mice to confirm whether Fyn is a valid pharmacological target of DAergic neurodegeneration. Primary mesencephalic neurons from Fyn KO mice were greatly protected from MPP+-induced DAergic cell death, neurite loss and DA reuptake loss. Furthermore, Fyn KO mice were significantly protected from MPTP-induced PKCδ-Y311 phosphorylation, behavioral deficits and nigral DAergic degeneration. This study thus unveils a mechanism by which Fyn regulates PKCδ′s pro-apoptotic function and DAergic degeneration. Pharmacological inhibitors directed at Fyn activation could prove to be a novel therapeutic target in the delay or halting of selective DAergic degeneration during PD.

Necrosis, apoptosis, necroptosis, three modes of action of dopaminergic neuron neurotoxins.

Most of the Parkinson’s disease (PD) cases are sporadic, although several genes are directly related to PD. Several pathways are central in PD pathogenesis: protein aggregation linked to proteasomal impairments, mitochondrial dysfunctions and impairment in dopamine (DA) release. Here we studied the close crossing of mitochondrial dysfunction and aggregation of α-synuclein (α-syn) and in the extension in the dopaminergic neuronal death. Here, using rat primary cultures of mesencephalic neurons, we induced the mitochondrial impairments using “DA-toxins” (MPP+, 6OHDA, rotenone). We showed that the DA-Toxins induced dopaminergic cell death through different pathways: caspase-dependent cell death for 6OHDA; MPP+ stimulated caspase-independent cell death, and rotenone activated both pathways. In addition, a decrease in energy production and/or a development of oxidative stress were observed and were linked to α-syn aggregation with generation of Lewy body-like inclusions (found inside and outside the dopaminergic neurons). We demonstrated that any of induced mitochondrial disturbances and processes of death led to α-syn protein aggregation and finally to cell death. Our study depicts the cell death mechanisms taking place in in vitro models of Parkinson’s disease and how mitochondrial dysfunctions is at the cross road of the pathologies of this disease.

Mechanistic Interplay Between Autophagy and Apoptotic Signaling in Endosulfan-Induced Dopaminergic Neurotoxicity: Relevance to the Adverse Outcome Pathway in Pesticide Neurotoxicity.

Chronic exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). Previously, we showed that dieldrin induces dopaminergic neurotoxicity by activating a cascade of apoptotic signaling pathways in experimental models of PD. Here, we systematically investigated endosulfan's effect on the interplay between apoptosis and autophagy in dopaminergic neuronal cell models of PD. Exposing N27 dopaminergic neuronal cells to endosulfan rapidly induced autophagy, indicated by an increased number of autophagosomes and LC3-II accumulation. Prolonged endosulfan exposure (>9 h) triggered apoptotic signaling, including caspase-2 and -3 activation and protein kinase C delta (PKCδ) proteolytic activation, ultimately leading to cell death, thus demonstrating that autophagy precedes apoptosis during endosulfan neurotoxicity. Furthermore, inhibiting autophagy with wortmannin, a phosphoinositide 3-kinase inhibitor, potentiated endosulfan-induced apoptosis, suggesting that autophagy is an early protective response against endosulfan. Additionally, Beclin-1, a major regulator of autophagy, was cleaved during the initiation of apoptotic cell death, and the cleavage was predominantly mediated by caspase-2. Also, caspase-2 and caspase-3 inhibitors effectively blocked endosulfan-induced apoptotic cell death. CRISPR/Cas9-based stable knockdown of PKCδ significantly attenuated endosulfan-induced caspase-3 activation, indicating that the kinase serves as a regulatory switch for apoptosis. Additional studies in primary mesencephalic neuronal cultures confirmed endosulfan's effect on autophagy and neuronal degeneration. Collectively, our results demonstrate that a functional interplay between autophagy and apoptosis dictate pesticide-induced neurodegenerative processes in dopaminergic neuronal cells. Our study provides insight into cell death mechanisms in environmentally linked neurodegenerative diseases.

The novel protective effects of loganin against 1-methyl-4-phenylpyridinium-induced neurotoxicity: Enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway.

Loganin, a major iridoid glycoside obtained from fruits of Cornus officinalis, possesses anti-inflammatory, antitumor, antidiabetic, and osteoporosis prevention effects. Loganin has been linked to neuroprotection in several models of neurodegeneration, including Parkinson's disease (PD). However, mechanisms underlying the neuroprotective effects of loganin are still mostly unknown. Here, we demonstrated the protective effects of loganin against PD mimetic toxin 1-methyl-4-phenylpyridinium (MPP+ ) and the important roles of insulin-like growth factor 1 receptor (IGF-1R) and glucagon-like peptide 1 receptor (GLP-1R) in the neuroprotective mechanisms of loganin. In primary mesencephalic neuronal cultures treated with or without MPP+ , loganin up-regulated expressions of neurotrophic signals including IGF-1R, GLP-1R, p-Akt, BDNF, and tyrosine hydroxylase. Loganin protected against MPP+ -induced apoptosis by up-regulating antiapoptotic protein and down-regulating proapoptotic protein. Moreover, loganin attenuated MPP+ -induced neurite damage via up-regulation of GAP43 and down-regulation of membrane-RhoA/ROCK2/p-LIMK/p-cofilin. Loganin also attenuated MPP+ -induced reactive oxygen species (ROS) production. However, both AG1024, an IGF-1R antagonist, and exendin 9-39, a GLP-1R antagonist, attenuated the protective effects of loganin on MPP+ -induced cytotoxicity, apoptosis, neurite length decrease, and ROS production. Our results suggest that loganin attenuates MPP+ -induced apoptotic death, neurite damage, and oxidative stress through enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway, providing the evidence that loganin possesses novel neuroprotective effects.

Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP+) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP+ reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP+-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP+-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP+-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson's toxin through enhancing antioxidative defense and preventing apoptotic death

Liuwei dihuang (LWDH), a widely used traditional Chinese medicine (TCM), has been employed as an anti-aging prescription to improve declined function. Parkinson's disease (PD) is a common adult-onset neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons with complex pathological mechanisms, including oxidative stress. Increasing evidence indicate that TCM has the potential to be neuroprotective drugs because of their antioxidant characteristics. The aim of this study is to investigate the mechanisms of LWDH-mediated protection in Parkinson's toxin-induced dopaminergic neurodegeneration by evaluating water extract of LWDH (LWDH-WE) in 1-methyl-4-phenylpyridinium (MPP+)-treated primary mesencephalic neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. In the present study, chemical profiling and quantitative analysis of LWDH-WE were revealed using 3D-HPLC technique, and were confirmed by the data of three batches of LWDH-WE. In primary mesencephalic neuronal cultures, LWDH-WE decreased MPP+-induced loss of tyrosine hydroxylase (TH)-positive neurons and increase of Annexin V-positive neurons. LWDH-WE reduced MPP+-induced oxidative damage via increasing antioxidant defense (SOD, GSH), decreasing ROS production, and down-regulating NADPH oxidases (Nox2 and Nox4). Also, LWDH-WE inhibited neuronal apoptosis by improving mitochondrial membrane potential, increasing antiapoptotic protein Bcl-2 expression, and down-regulating apoptotic signaling (Bax, cytochrome c, cleaved-caspase-3) in MPP+-treated neurons. In MPTP-treated C57BL/6 mice, LWDH-WE attenuated TH-positive neuronal loss in substantia nigra pars compacta (SNpc), and improved locomotor activity of mice. In conclusion, the present results reveal that LWDH-WE possesses protection on dopaminergic neurons through enhancing antioxidant defense and decreasing apoptotic death, suggesting the potential benefits of LWDH-WE for PD treatment.

Lipid-like components released from degenerating dopaminergic neurons trigger the dynamic migration of microglia

In the brain, communication between neural and non-neural cells is crucial for the proper functioning of the central nervous system. Microglia play an important role in the clearance of neural cellular corpses and debris, especially under pathological conditions. It remains, however, unclear how microglia sense the degenerating neurons at a distance in order to migrate to them. In the present study, we explored the interaction between neurons and microglia using an in vitro model of Parkinson’s disease (PD). In primary mesencephalic neuronal cultures, 1-methyl-4-phenylpridinium (MPP+) induced the selective death of dopaminergic (DAergic) neurons in a dose- and time-dependent manner. Transmigration assay showed that the conditioned medium (CM) from mesencephalic cultures treated with MPP+ was enough to trigger the attraction of microglia at an early as well as a late phase of neuronal damage. Microglia preferably reacted with the soluble parts separated by ultracentrifugation over the neural debris-containing pellets. This chemoattractive activity was significantly reduced by the removal of the lipidic components in CM, but not by the removal of proteins, DNA or RNA. These results suggest that as yet-unidentified lipid-like components released from dying DAergic neurons are likely to recruit microglia, and thus have a role in neuronal damage.

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation.

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson's disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.

Wnt5a-Dopamine D2 Receptor Interactions Regulate Dopamine Neuron Development via Extracellular Signal-regulated Kinase (ERK) Activation

The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R(-/-)) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R(-/-) mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R(-/-) mice. Co-immunoprecipitation and displacement of [(3)H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway.

Human dental pulp stem cells protect mouse dopaminergic neurons against MPP+ or rotenone

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive death of substantia nigra dopaminergic neurons that results in a regional loss of striatal dopamine (DA) levels. Dental pulp contains ex vivo-expandable cells called dental pulp stem cells (DPSCs), with the capacity to differentiate into multiple cell lineages. More interestingly, due to their embryonic origin, DPSCs express neurotrophic factors such as brain-derived neurotrophic factor, nerve growth factor and glial cell-derived neurotrophic factor. The aim of the present study was to investigate the neuroprotective effects of DPSCs against MPP+ (2.5, 5, and 10μM) and rotenone (0.25, 0.5 and 1μM) in an in vitro model of PD, using an indirect co-culture system with mesencephalic cell cultures. When mesencephalic cultures were challenged with MPP+ or rotenone, in the presence of DPSCs a statistically significant protective effect was observed at all the tested doses in terms of DA uptake. DPSCs protective effect on DA neurons was also confirmed by immunocytochemistry: an increased number of spared tyrosine hydroxylase (TH)+ cells was observed in co-culture conditions compared to controls, and neurons showed longer processes in comparison with mesencephalic cells grown without DPSCs. In conclusion, the co-culture with DPSCs significantly attenuated MPP+ or rotenone-induced toxicity in primary cultures of mesencephalic neurons. Considering that the direct contact between the two cell types was prevented, it can be speculated that neuroprotection could be due to soluble factors such as BDNF and NGF, released by DPSCs. Blocking BDNF and NGF with neutralizing antibodies, the neuroprotecting effect of DPSCs was completely abolished. Therefore DPSCs can be viewed as possible candidates for studies on cell-based therapy in neurodegenerative disorders.

PKCβ co-localizes with the dopamine transporter in mesencephalic neurons

The dopamine transporter (DAT) is a critical regulator of dopaminergic neurotransmission. Research in both rat striatum and heterologous cells suggests that protein kinase C beta (PKCβ) is important for proper trafficking of DAT. However, a critical gap that is missing from the literature is the localization of PKCβ to mesencephalic dopaminergic neurons. In this study we examined the co-localization of DAT, which serves to identify dopaminergic neurons, and PKCβ in mesencephalic dopaminergic cells. Using immunofluorescence and confocal microscopy, we demonstrated co-localization of DAT and PKCβ in primary cultures of mesencephalic neurons and in dopamine neurons in rat substantia nigra and ventral tegmental area. PKCβ was not specific for dopamine neurons in the two brain regions. This is the first demonstration of co-localization of PKCβ and DAT in mesencephalic neurons. The co-localization of PKCβ with DAT in mesencephalic neurons corroborates our previous studies demonstrating a role for PKCβ in DAT function.

Environmental Neurotoxic Pesticide Increases Histone Acetylation to Promote Apoptosis in Dopaminergic Neuronal Cells: Relevance to Epigenetic Mechanisms of Neurodegeneration

Pesticide exposure has been implicated in the etiopathogenesis of Parkinson's disease (PD); in particular, the organochlorine insecticide dieldrin is believed to be associated with PD. Emerging evidence indicates that histone modifications play a critical role in cell death. In this study, we examined the effects of dieldrin treatment on histone acetylation and its role in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. In mesencephalic dopaminergic neuronal cells, dieldrin induced a time-dependent increase in the acetylation of core histones H3 and H4. Histone acetylation occurred within 10 min of dieldrin exposure indicating that acetylation is an early event in dieldrin neurotoxicity. The hyperacetylation was attributed to dieldrin-induced proteasomal dysfunction, resulting in accumulation of a key histone acetyltransferase (HAT), cAMP response element-binding protein. The novel HAT inhibitor anacardic acid significantly attenuated dieldrin-induced histone acetylation, Protein kinase C delta proteolytic activation and DNA fragmentation in dopaminergic cells protected against dopaminergic neuronal degeneration in primary mesencephalic neuronal cultures. Furthermore, 30-day exposure of dieldrin in mouse models induced histone hyperacetylation in the striatum and substantia nigra. For the first time, our results collectively demonstrate that exposure to the neurotoxic pesticide dieldrin induces acetylation of core histones because of proteasomal dysfunction and that hyperacetylation plays a key role in dopaminergic neuronal degeneration after exposure of dieldrin.

Oxidative Modification of Peroxiredoxin Is Associated with Drug-induced Apoptotic Signaling in Experimental Models of Parkinson Disease

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.

Leptin Protects against 6-Hydroxydopamine-induced Dopaminergic Cell Death via Mitogen-activated Protein Kinase Signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1 μM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24–48 h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI 3K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.

Neuroprotective Effect of Protein Kinase Cδ Inhibitor Rottlerin in Cell Culture and Animal Models of Parkinson's Disease

Recent studies from our laboratory demonstrated that the protein kinase C (PKC) delta isoform is an oxidative stress-sensitive kinase and a key mediator of apoptotic cell death in Parkinson's Disease (PD) models (Eur J Neurosci 18:1387-1401, 2003; Mol Cell Neurosci 25:406-421, 2004). We showed that native PKC delta is proteolytically activated by caspase-3 and that suppression of PKC delta by dominant-negative mutant or small interfering RNA against the kinase can effectively block apoptotic cell death in cellular models of PD. In an attempt to translate the mechanistic studies to a neuroprotective strategy targeting PKC delta, we systematically characterized the neuroprotective effect of a PKC delta inhibitor, rottlerin, in 1-methyl-4-phenylpyridinium (MPP(+))-treated primary mesencephalic neuronal cultures as well as in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. Rottlerin treatment in primary mesencephalic cultures significantly attenuated MPP(+)-induced tyrosine hydroxylase (TH)-positive neuronal cell and neurite loss. Administration of rottlerin, either intraperitoneally or orally, to C57 black mice showed significant protection against MPTP-induced locomotor deficits and striatal depletion of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. Notably, rottlerin post-treatment was effective even when MPTP-induced depletion of dopamine and its metabolites was greater than 60%, demonstrating its neurorescue potential. Furthermore, the dose of rottlerin used in neuroprotective studies effectively attenuated the MPTP-induced PKC delta kinase activity. Importantly, stereological analysis of nigral neurons revealed rottlerin treatment significantly protected against MPTP-induced TH-positive neuronal loss in the substantia nigra compacta. Collectively, our findings demonstrate the neuroprotective effect of rottlerin in both cell culture and preclinical animal models of PD, and they suggest that pharmacological modulation of PKC delta may offer a novel therapeutic strategy for treatment of PD.

GDNF abates serum deprivation-induced tyrosine hydroxylase Ser19 phosphorylation and activity

High dopamine levels can contribute to neuronal dysfunction, impair plasticity and be toxic to neuronal cells in pathological conditions. The synthesis of dopamine is regulated by phosphorylation of the rate-limiting enzyme tyrosine hydroxylase (TH) under physiological conditions, with the phosphorylation of Ser31 and Ser40 directly increasing TH activity. Although a third phosphorylation site, Ser19, does not appear to directly regulate TH activity in physiological conditions, its role in pathological conditions is poorly understood. In this study, we examined the effects of serum deprivation (to mimic loss of retrogradely/anterogradely transported target-derived neurotrophic factors following axonal injury) and glutamate receptor stimulation (to mimic excitotoxicity) on TH phosphorylation and activity in a cell line and in mesencephalic primary culture cells. In addition, we also tested whether glial cell line-derived neurotrophic factor (GDNF) can alter these changes. We demonstrate that serum-deprivation resulted in a sustained increase in Ser19 phosphorylation beginning at 3 h and lasting up to 10 h without any detectable change in Ser31 or Ser40 phosphorylation within this time frame. This increase in Ser19 phosphorylation was associated with enhanced TH activity and was due, in part, to glutamate-receptor-mediated calcium influx and possibly calcium/calmodulin-dependent protein kinase II (CaMKII) activation. Interestingly in this serum-deprivation model, GDNF blocked the increase in Ser19 phosphorylation and TH activity at the 10-h time point following serum deprivation. Furthermore, GDNF also blocked the glutamate-mediated increase in Ser19 phosphorylation in rat primary mesencephalic neuronal cultures. Taken together, these findings suggest that GDNF may reduce dopamine synthesis in pathological conditions.

Centrosome overduplication induced by rotenone treatment affects the cellular distribution of p53 tumor suppressor protein in the neuroblastoma B65 cell line

Abstract Recently, the formation of inclusion bodies in several neurodegenerative diseases, including Parkinson's disease, has been associated with the aggregation of unfolded proteins recruited in the centrosome. We have reported previously that rotenone, an insecticide that is used to produce experimental models of Parkinsonism, induced the aggregation of the α‐synuclein protein in the centrosome, and it notably affected the structure and function of this organelle in primary cultures of mesencephalic neurons and astrocytes. However, it is still obscure the mechanisms through which the disorganization and centrosomal dysfunction could induce cell death. In this study the rat neuroblastoma B65 cell line was chronically exposed to rotenone, and then the distribution of the centrosomal protein γ‐tubulin was studied by immunocytochemistry and Western blot analyses. Finally, the configuration of mitotic spindles and distribution of the p53 protein was observed in the control and rotenone‐treated groups. Rotenone treatment increased the number of cells having centrosome overduplication and multipolar mitotic spindles. In contrast, rotenone induced redistribution of the p53 protein, which was colocalized with the γ‐tubulin protein in the perinuclear region of cells having overduplicated centrosomes. In addition, the p53 positive signal was markedly intense in cells containing aberrant chromosome segregation and micronuclei. Our results suggest that centrosome overduplication may play an important role in the redistribution of the p53 protein in rotenone‐treated cells, and this could represent an alternative mechanism of rotenone to induce apoptosis in neuronal cells.

Neurotoxic Calcium Transfer from Endoplasmic Reticulum to Mitochondria Is Regulated by Cyclin-Dependent Kinase 5-Dependent Phosphorylation of Tau

Increased levels of mitochondrial-free calcium have been associated with several cell-death paradigms, such as excitotoxicity and ceramide-mediated neuronal death. In the latter, calcium is transferred from the endoplasmic reticulum to mitochondria by a mechanism that is only partly understood. We show here that CDK5 (cyclin-dependent kinase 5) plays a role. Free calcium levels in the endoplasmic reticulum and mitochondria were measured with fluorescent markers in C2-ceramide-treated primary cultures of mesencephalic neurons and differentiated pheochromocytoma PC12 cells. Calcium levels decreased in the endoplasmic reticulum as they increased in mitochondria. Both changes were blocked by the pharmacological and molecular CDK5 inhibitors roscovitine and a dominant-negative form of CDK5. Although the kinase did not mediate the transfer of calcium per se, which required the proapoptotic Bcl-2 family protein t-Bid (the truncated form of Bid), it facilitated the transfer by inducing the clustering of endoplasmic reticulum and mitochondria around the centrosome where they formed close contacts, as shown by immunocytochemistry and electron microscopy. Organelle clustering resulted from CDK5-dependent phosphorylation of the microtubule-associated protein tau on threonine 231. This caused its release from microtubules into the soluble fraction of cellular proteins, which appears to favor retrograde transport of the organelles. Mutation of threonine 231 to alanine, so that tau could not be phosphorylated at this site, prevented the ceramide-induced release of tau from microtubules, organelle clustering, the increase in mitochondrial-free calcium levels, and neuronal death, demonstrating the importance of the CDK5-dependent signaling cascade in this calcium-dependent cell-death mechanism.

Phosphorylation of p38 MAPK Induced by Oxidative Stress Is Linked to Activation of Both Caspase-8- and -9-mediated Apoptotic Pathways in Dopaminergic Neurons

We evaluated the contribution of p38 mitogen-activated protein kinase and the events upstream/downstream of p38 leading to dopaminergic neuronal death. We utilized MN9D cells and primary cultures of mesencephalic neurons treated with 6-hydroxydopamine. Phosphorylation of p38 preceded apoptosis and was sustained in 6-hydroxydopamine-treated MN9D cells. Co-treatment with PD169316 (an inhibitor of p38) or expression of a dominant negative p38 was neuroprotective in death induced by 6-hydroxydopamine. The superoxide dismutase mimetic and the nitric oxide chelator blocked 6-hydroxydopamine-induced phosphorylation of p38, suggesting a role for superoxide anion and nitric oxide in eliciting a neurotoxic signal by activating p38. Following 6-hydroxydopamine treatment, inhibition of p38 prevented both caspase-8- and -9-mediated apoptotic pathways as well as generation of truncated Bid. Consequently, 6-hydroxydopamine-induced cell death was rescued by blockading activation of caspase-8 and -9. In primary cultures of mesencephalic neurons, the phosphorylation of p38 similarly appeared in tyrosine hydroxylase-positive, dopaminergic neurons after 6-hydroxydopamine treatment. This neurotoxin-induced phosphorylation of p38 was inhibited in the presence of superoxide dismutase mimetic or nitric oxide chelator. Co-treatment with PD169316 deterred 6-hydroxydopamine-induced loss of dopaminergic neurons and activation of caspase-3 in these neurons. Furthermore, inhibition of caspase-8 and -9 significantly rescued 6-hydroxydopamine-induced loss of dopaminergic neurons. Taken together, our data suggest that superoxide anion and nitric oxide induced by 6-hydroxydopamine initiate the p38 signal pathway leading to activation of both mitochondrial and extramitochondrial apoptotic pathways in our culture models of Parkinson's disease.