Development of a method to knock down HS3ST2 expression in a cellular model of tauopathy

VERA, CECILIA; TROTIER, ALEXANDRE; OUIDJA, MOHAND OUIDIR; FARIAS, RN

Congreso; 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology (SAIB); 2015

Tau abnormal phosphorylation has been identified as a critical event characterizing tauopathies such as Alzheimer?sdisease (AD) and frontotemporal dementias. It seems to require polyanions such as heparin. Furthermore, recentfindings show that heparan sulfates (HS) can induce Tau abnormal phosphorylation in cellular models of tauopathy,were they colocalize and interact with Tau. Moreover, the enzyme HS3ST2, responsible for the 3-O-sulfation of HS,is overexpressed in AD brains. All this raises the question whether HS3ST2, or its products, are involved in thedevelopment of Tau-mediated neurodegeneration. In this work we aim to set up a method to knock down HS3ST2expression in a mutation-dependent cellular model of tauopathy, consisting of differentiated SH-SY5Y humanneuroblastoma cell line expressing mutant P301L Tau. To achieve it, we transducted cells with a lentiviral vectorencoding shRNA sequences designed to induce HS3ST2 mRNA degradation. Transduction efficiency was assessedby fluorescence microscopy and flow cytometry. A protocol for qPCR and western blot determination of knock downefficiency was designed. We also characterized Tau and HS cell location in both normal and tauopathy conditions byconfocal microscopy. Our results indicate that inhibiting HS3ST2 gene expression is possible with conditionsoptimization