6-Hydroxydopamine decreases brain aldehyde dehydrogenase 2 (ALDH2) expression: Im-plications for neurotoxicity in a Parkinson´s disease model”.

DEZA PONZIO ROMINA; HERRERA MACARENA; MARCHESSE NATALIA; BASDMAJIAN, MARTIN; BELLINI MARIA J; MOLINA VICTOR; VIRGOLINI MB,; HERENU CLARUDIA

Congreso; Joint Meeting Neurotoxicity Society and International Neurotoxicology Association.; 2017

“6-Hydroxydopamine decreases brain aldehyde dehydrogenase 2 (ALDH2) expression: Implications for neurotoxicity in a Parkinson´s disease model”Romina Deza-Ponzio, Macarena L. Herrera, Natalia A. Marchese, Osvaldo M. Basmadjian, María José Bellini, Víctor A. Molina, Miriam B. Virgolini and Claudia B. Hereñú.Parkinson’s disease (PD) is one of the most common neurodegenerative diseases characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Among the different hypothesis related to PD pathogenesis, the mitochondrial enzyme ALDH2 is of particular interest due to its crucial role in the disposition of neurotoxic metabolites such as DOPAL and DOPEGAL in the monoamine neurotransmitter pathway.. Thus, the aim of this study was examine ALDH2 expression in the dopaminergic nigroestriatal pathway in a 6-hydroxydopamine (6-OHDA) animal model of PD. In addition, although motor symptoms are the main clinical features of PD, there is increasing evidence showing that PD patients also develop cognitive dysfunctions. Hence, a second approach sought to evaluate cognitive parameters in the same model.Animals and treatment: Male adult Wistar rats (280 - 320 g) bred and raised at the Facultad de Ciencias Químicas vivarium were anesthetized with Ketamine and Xilazine (in a 2/3 ratio) and mounted into a stereotaxic instrument (David Kopf, USA) with the incisor bar at 3.3 cm above the interaural line. Afterwards, animals were bilaterally microinjected in dorsal striatum (CPu) according with the coordinates from Paxinos and Watson (2009) (AP: +0.2 mm, ML: ±3.5mm, DV: -4.8 mm) with either vehicle (saline solution with 0.02% ascorbic acid, SHAM group) or 2 uL of a dose of 10 ug/ul 6-OHDA at the rate of 0.5 uL/min (6-OHDA group). After each injection, the needle (22G, 12mm length) was left in place for 2–4 min to minimize backflow of the solution. All procedures were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee of the Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Argentina.Experimental design and behavioral tests: Twenty days after the lesion and before the animals were sacrificed; they were tested for short-term spatial memory with a modified version of Y-maze test (Matheus et al. 2016). The Y-maze apparatus consisted of three arms (50 cm long, 10 cm wide and 20 cm high). The task consisted of two trials (training and test) of 5 min each separated by an inter-trial interval of 120 min. During the training trial, with one arm labeled ‘novel’ blocked by a removable door the rats were placed on the intersection of the ‘start’ arm facing the center and were given the possibility to choose between the ‘start’ and the remaining arm. During the test trial with the ‘novel’ arm left opened the percentage of entries and the percentage of time spent in the ‘novel’ arm were recorded. Entry into an arm was defined as placement of all four paws into the arm. Results were assessed by 2-way ANOVA and Bonferroni´s pos hoc test.Fixed brains were sectioned into 40-um-thick sections in series of six. Free-floating sections were washed with phosphate-buffered saline (PBS) and immunohistochemistry was performed for TH and ALDH2 in CPu, SN, dorsal hippocampus (CA1) and prefrontal cortex (PFC). The sections were incubated overnight in mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (1:200; Millipore) or mouse monoclonal Anti ALDH2; antibody (1:1000, Abcam) at 4°C, followed by incubation in biotinylated secondary antibody (1:2000; anti-mouse or 1:1000 anti-rabbit; Jackson Immunoresearch or Abcam, respectively). The signal was visualized with diaminobenzidine (DAB) as a chromogen. All DAB-stained slides were imaged in an optical microscope (Leica) using a 20X objective. Results were expressed as Density Units (optical density) and percentage of positive cells using ImageJ software.All data were compared by Student´s t-test (p<0.05 considered as statistically significant). CONCLUSION: Our results show a reduction in both, TH and ALDH2 immunostaining. They suggest a catecholaldehyde basis for the neurotoxicity and neurodegeneration characteristic of the 6-OHDA model. Whether the cognitive dysfunction that we observed in this experimental animal model of PD Is related to toxic DA metabolites buildup is a topic that deserves further research.