APOE4 HETEROLOGOUS EXPRESSION, PURIFI- CATION UNDER NON-DENATURING CONDITIONS AND EFFECTS ON THE MITOCHONDRIAL NETWORK OF A NEURONAL CLONAL CELL LINE

EZEQUIEL A. SERRANO; ANGEL GABRIEL VALDIVIESO; PABLO G. SANZ; GUILLERMO L. TAMINELLI; FRANCISCO J. BARRANTES

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAI&FAIC SAFIS 2022; 2022

SAIC SAI&FAIC SAFIS

Apolipoprotein E (ApoE) is a major risk factor for Alzheimer disease.The APOE4 allele increases the risk up to 15 times in homozygouscarriers. To learn about the effects of ApoE4 on neuronal cells, wepurified the recombinant protein expressed in the E. Coli BL21 strain.Soluble ApoE4 was purified by affinity chromatography using a Ni-NTA resin, followed by the release of the His-Trx tag with 3C-pro-tease and size exclusion chromatography. A yield of ~10 mg/L wasobtained with a purity of 95%, as analyzed by SDS polyacrylamidegel electrophoresis. The structural integrity and biological functionof the purified ApoE4 was analyzed by circular dichroism and aDMPC assay. To analyze the possible effect of ApoE4 on mitochon-drial function, samples of the clonal cell line CNh were incubatedwith 17 mg/mL of recombinant ApoE4 for 24 h. Mitochondria werelabeled with MitoTracker Orange and imaged in vivo. Mitochondrialmorphology was analyzed with MiNA and MicroP. Reactive oxygenspecies (ROS) and mitochondrial membrane potential (Ψm) werestudied with the fluorescent probes DCFH-DA and TMRE, respec-tively. Both MiNa and MicroP analyses showed that ApoE4 mod-ulates the mitochondrial network, with an increase in mitochondri-al fusion and proliferation. The Ψm decreased upon treatment ofCNh cells with ApoE4 (p