EXPRESSION, PURIFICATION OF RECOMBINANT APOLIPOPROTEIN E4 UNDER NON-DENATURING CONDITIONS, AND MITOCHONDRIAL MORPHOLOGY ANALYSIS IN A NEURONAL CLONAL CELL LINE

VALDIVIESO, GABRIEL; SERRANO, E.A.; BARRANTES, FRANCISCO J.

Buenos Aires

Congreso; Reunion Anual SAIC, Soc Nanomedicina; 2021

SAIC

Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease. The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects, ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, we aimed at purifying the recombinant protein expressed in E. coli and assaying it on mitochondria of a neuronal clonal cell line, CNh. E. coli BL21 strain expressing the ApoE4 (pET32-E43C, containing His and Trx-tags) and 3C-protease were grown in LB medium and induced with 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively. Crude soluble ApoE was purified by affinity chromatography using a Ni-NTA resin. The 3C-protease was purified by FPLC (Superose-12 size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a 25:1 ratio to release the His-Trx tag. ApoE4 was next purified by size exclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDS polyacrylamide gels. We next studied mitochondrial morphology in neuronal CNh cells using confocal fluorescence microscopy. Cells were incubated with17mg/mL ApoE4 and mitochondria labelled with MitoTracker Orange and imaged in vivo. Mitochondrial morphology was analyzed with the Mitochondrial Network Analysis plugin for Fiji. Individual counts (1 branch), network length and mitochondrial footprint (area covered) were quantified. ApoE4 induced an increase in mitochondrial footprint (p