Apoptosis In Dopaminergic Cells Research Articles

Oxidative stress and mitochondrial-mediated apoptosis in dopaminergic cells exposed to methylcyclopentadienyl manganese tricarbonyl.

Methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing gasoline additive, was investigated to determine whether MMT potentially causes dopaminergic neurotoxic effects. MMT is acutely cytotoxic and dopamine-producing cells (PC-12) seemed to be more susceptible to cytotoxic effects than nondopaminergic cells (striatal gamma-aminobutyric acidergic and cerebellar granule cells). MMT also potently depleted dopamine apparently by cytoplasmic vesicular release to the cytosol, a neurochemical change resembling other dopaminergic neurotoxicants. Generation of reactive oxygen species (ROS), an early effect in toxicant-induced apoptosis, occurred within 15 min of MMT exposure. MMT caused a loss of mitochondrial transmembrane potential (DeltaPsim), a likely source of ROS generation. The ROS signal further activated caspase-3, an important effector caspase, which could be inhibited by antioxidants (Trolox or N-acetyl cysteine). Predepletion of dopamine by using alpha-methyl-p-tyrosine (tyrosine hydroxylase inhibitor) treatment partially prevented caspase-3 activation, denoting a significant dopamine and/or dopamine by-product contribution to initiation of apoptosis. Genomic DNA fragmentation, a terminal hallmark of apoptosis, was induced concentration dependently by MMT but completely prevented by pretreatment with Trolox, deprenyl (monoamine oxidase-B inhibitor), and alpha-methyl-p-tyrosine. A final set of critical experiments was performed to verify the pharmacological studies using a stable Bcl-2-overexpressing PC-12 cell line. Bcl-2-overexpressing cells were significantly refractory to MMT-induced ROS generation, caspase-3 activation, and loss of DeltaPsim and were completely resistant to MMT-induced DNA fragmentation. Taken together, the results presented herein demonstrate that oxidative stress plays an important role in mitochondrial-mediated apoptotic cell death in cultured dopamine-producing cells after exposure to MMT.

A novel oxidative stress dependent apoptotic pathway in pesticide‐induced dopaminergic degeneration in PD models

Emerging epidemiological and case control studies suggest that environmental factors especially pesticides are dominant risk factors in the etiology of idiopathic Parkinson's disease (PD). In the present study, we characterized oxidative stress dependent cellular events by exposing rat mesencephalic dopaminergic cell line (N27) and PC 12 cells to dieldrin (a potential environmental risk factor for development of PD). Exposure of dieldrin (30–200 μm) resulted in a rapid increase in generation of reactive oxygen species within 30 min and was followed by release of mitochondrial cytochrome‐c into cytoplasm. Cytochrome‐c then activated a proapoptotic cysteine protease, caspase‐3 in a time‐ and dose‐dependent manner. Interestingly, we also found that dieldrin exposure induces a time‐ and dose‐dependent proteolytic cleavage of native protein kinase Cδ (PKCδ, 74 kDa) to yield 41‐kDa catalytically active and 38‐kDa regulatory fragments. Pretreatment with caspase inhibitors blocked dieldrin‐induced proteolytic cleavage of PKCδ, indicating that cleavage is mediated by caspase‐3. Attenuation of ROS generation, caspase‐3 activation or overexpression of mutant PKCδK376R (dominant negative) almost completely suppressed dieldrin‐induced DNA fragmentation. We also found that dieldrin exposure activates the redox sensitive transcription factor NF‐kB through PKCδ dependent manner. Together, these data demonstrate that caspase‐3 dependent proteolytic activation of PKCδ plays a key role in oxidative stress‐mediated apoptosis in dopaminergic cells following exposure to an environmental neurotoxic agent.Acknowledgements: Supported in part by ES 10586.

Caspase-3-Dependent Proteolytic Cleavage of Protein Kinase Cδ Is Essential for Oxidative Stress-Mediated Dopaminergic Cell Death after Exposure to Methylcyclopentadienyl Manganese Tricarbonyl

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5--15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCdelta, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCdelta activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCdelta cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCdelta. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCdelta fragment. Attenuation of ROS generation, caspase-3 activation, and PKCdelta activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCdelta(K376R) (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.

Preclinical (premotor) Parkinson’s disease

Parkinson's disease (PD) is an idiopathic disease caused by necrosis and apoptosis of dopaminergic cells in the brainstem, which are probably induced by oxidative stress. Current therapeutic strategies comprise symptomatic and restorative treatment. Neuroprotective treatment, however, is close to becoming reality. As neuroprotective therapy may be of particular benefit to the preclinical and/or very early PD patients, identifying patients in the early stages of the disease is a priority. Both [18F]dopa positron emission tomography (PET) and [123I]beta-CIT single photon emission computed tomography (SPECT) imaging may be useful tools in diagnosing early (preclinical) PD. As screening the whole population for preclinical PD is not realistic, one has to select subjects with a high risk for this disease. Olfactory disturbances, subtle neurocognitive dysfunction, visuomotor control abnormalities and, to a lesser degree, mood and personality disorders, have lately been suggested to precede or accompany early clinical motor hallmarks of PD. In an epidemiological study, 500 first-degree relatives of PD patients were assessed for these signs and symptoms, and [123I]beta-CIT SPECT was performed on patients in the top 10% and the bottom 10% with regard to sense of smell. In this report, the study design and initial data from this ongoing study will be presented.

6-Hydroxydopamine induced apoptosis of dopaminergic cells in the rat substantia nigra.

The pathologic hallmark of Parkinson's disease is the dopaminergic cell death in the substantia nigra (SN). The cause of the cell death is, however, unknown. Even the question on whether the cells die by apoptosis or necrosis has not been answered with certainty. In 6-Hydroxydopamine induced Parkinsonian rats, the present study observed apoptotic nuclei from 1 day to 14 days after lesioning, using the TdT(terminal deoxynucleotidyl transferase)-mediated dUTP-biotin nick-end labeling method. Tyrosine hydroxylase immunohistochemistry and haematoxylin staining further revealed that these apoptotic cells are dopaminergic cells in the substantia nigra. The results suggest that dopaminergic cells in SN undergo apoptosis in the rat model of Parkinson's disease.