The Catecholaminergic RCSN-3 Cell Line: A Model to Study Dopamine Metabolism

RCSN-3 cells are a cloned cell line derived from the substantia nigra of an adult rat. The cell line grows in monolayer and does not require differentiation to express catecholaminergic traits, such as (i) tyrosine hydroxylase; (ii) dopamine release; (iii) dopamine transport; (iv) norepinephrine transport; (v) monoamine oxidase (MAO)-A expression, but not MAO-B; (vi) formation of neuromelanin; (vii) vesicular monoamine transporter-2 (VMAT-2) expression. In addition, this cell line expresses serotonin transporters, divalent metal transporter, DMT1, dopamine receptor 1 mRNA under proliferating conditions, and dopamine receptor 5 mRNA after incubation with dopamine or dicoumarol. Expression of dopamine receptors D2, D3 and D4 mRNA were not detected in proliferating cells or when the cells were treated with dopamine, CuSO4, dicoumarol or dopamine-copper complex. Angiotensin II receptor mRNA was also found to be expressed, but it underwent down regulation in the presence of aminochrome. Total quinone reductase activity corresponded 94% to DT-diaphorase. The cells also express antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. This cell line is a suitable in vitro model for studies of dopamine metabolism, since under proliferating conditions the cells express all the pertinent markers

Lrrk2 p.Q1111H substitution and Parkinson's disease in Latin America

Fil: Mata, Ignacio F. Veterans Affairs Puget Sound Health Care System, Seattle, Washington; Estados Unidos.Fil: Wilhoite, Greggory J. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Yearout, Dora. Veterans Affairs Puget Sound Health Care System, Seattle, Washington; Estados Unidos. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Bacon, Justin A. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Cornejo-Olivas, Mario. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Mazzetti, Pilar. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Marca, Victoria. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Ortega, Olimpio. Universidad Nacional Mayor de San Marcos. School of Medicine, Lima; Perú.Fil: Acosta, Oscar. Instituto Nacional de Ciencias Neurológicas. Movement disorders, Lima; Perú.Fil: Cosentino, Carlos. Instituto Nacional de Ciencias Neurológicas. Movement disorders, Lima; Perú.Fil: Torres, Luis. Universidad Nacional del Altiplano, Puno; Perú.Fil: Medina, Angel C. University of Chile. Faculty of Medicine. ICBM. Molecular and Clinical Pharmacology, Santiago; Chile.Fil: Perez-Pastene, Carolina. University of Chile. Faculty of Medicine. ICBM. Molecular and Clinical Pharmacology, Santiago; Chile.Fil: Díaz-Grez, Fernando. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Vilariño-Güell, Carles. Liga del Parkinson de Chile; Chile.Fil: Venegas, Pablo. Liga del Parkinson de Chile; Chile.Fil: Miranda, Marcelo. Liga del Parkinson de Chile; Chile.Fil: Trujillo-Godoy, Osvaldo. Hospital Barros Luco Trudeau; Chile.Fil: Layson, Luis. Hospital Barros Luco Trudeau; Chile.Fil: Avello, Rodrigo. Hospital Regional de Concepción; Chile.Fil: Dieguez, Elena. Universidad de la República. Facultad de Medicina. Departamento de Neurología, Montevideo; Uruguay.Fil: Raggio, Victor. Universidad de la República. Facultad de Medicina. Departamento de Genética, Montevideo; Uruguay.Fil: Micheli, Federico E. ANLIS Dr.C.G.Malbrán; Argentina.Fil: Perandones, Claudia. ANLIS Dr.C.G.Malbrán. Dirección Científico Técnica; Argentina.Fil: Alvarez, Victoria. Hospital Universitario Central de Asturias. Instituto de Investigación Nefrológica (IRSINFRIAT). Laboratorio de Genética Molecular, Oviedo; España.Fil: Segura-Aguilar, Juan. Instituto Nacional de Ciencias Neurológicas. Unidad de Neurogenética, Lima; Perú.Fil: Farrer, Matthew J. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Fil: Zabetian, Cyrus P. Veterans Affairs Puget Sound Health Care System, Seattle, Washington; Estados Unidos.Fil: Ross, Owen A. Mayo Clinic College of Medicine. Department of Neuroscience, Jacksonville, Florida; Estados Unidos.Mutations in the LRRK2 gene are the most common genetic cause of Parkinson's disease, with frequencies displaying a high degree of population-specificity. Although more than 100 coding substitutions have been identified, only seven have been proven to be highly penetrant pathogenic mutations. Studies however are lacking in non-white populations. Recently, Lrrk2 p.Q1111H (rs78365431) was identified in two affected Hispanic brothers and absent in 386 non-Hispanic white healthy controls. We therefore screened this variant in 1460 individuals (1150 PD patients and 310 healthy controls) from 4 Latin American countries (Peru, Chile, Uruguay and Argentina). In our case-control series from Peru and Chile we observed an increased frequency of Lrrk2 p.Q1111H in patients (7.9%) compared to controls (5.4%) although the difference did not reach significance (OR 1.38; p = 0.10). In addition, the frequency of Lrrk2 p.Q1111H varied greatly between populations and further screening in a set of pure Amerindian and pure Spanish controls suggested that this variant likely originated in an Amerindian population. Further studies in other Latin American populations are warranted to assess its role as a risk factor for Parkinson's disease. Screening in Parkinson's disease patients from under-represented populations will increase our understanding of the role of LRRK2 variants in disease risk worldwide

Copper·Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death*

Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less