Vitamin B12-Impaired Metabolism Produces Apoptosis and Parkinson Phenotype in Rats Expressing the Transcobalamin-Oleosin Chimera in Substantia Nigra

International audienceThe development of fearfulness and the capacity of animals to cope with stressful events are particularly sensitive to early experience with mothers in a wide range of species. However, intrinsic characteristics of young animals can modulate maternal influence. This study evaluated the effect of intrinsic fearfulness on non-genetic maternal influence. Quail chicks, divergently selected for either higher (LTI) or lower fearfulness (STI) and from a control line (C), were cross-fostered by LTI or STI mothers. Behavioural tests estimated the chicks' emotional profiles after separation from the mother. Whatever their genotype, the fearfulness of chicks adopted by LTI mothers was higher than that of chicks adopted by STI mothers. However, genetic background affected the strength of maternal effects: the least emotional chicks (STI) were the least affected by early experience with mothers. We demonstrated that young animal's intrinsic fearfulness affects strongly their sensitivity to non-genetic maternal influences. A young animal's behavioural characteristics play a fundamental role in its own behavioural development processes

Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

BACKGROUND: Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin. METHODOLOGY: chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells. PRINCIPAL FINDINGS: vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration. CONCLUSIONS/SIGNIFICANCE: the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest

Expression of transcobalamin/oleosin chimeric proteins in transfected N1E-115 cells.

<p>A: Transgenic expression in N1E-115 cells at 48 h after transfection using lipofectamine. The cells were transfected with one of the following plasmids: pCMV-TCII-OLEO coding for transcobalamin-oleosin (lane 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (lane 2), pCMV-TCII coding for transcobalamin II (lane 3), pCMV-OLEO coding for oleosin (lane 4), pCDNA3 (lane 5), and pCMV-GFP-TCII-OLEO coding for GFP-TCII-OLEO (lane 6). The housekeeping gene was β-actin. The size in bp of the amplified products was 1347 for transcobalamin-oleosin (TCII-OLEO), 1240 for oleosin-transcobalamin (OLEO-TCII), 551 for transcobalamin II (TCII), 275 for oleosin (OLEO), and 349 for β-actin. B: Western blotting of homogenate of N1E-115 cells transfected with the various plasmids. From lane 1 to 6: homogenates from cells transfected with pCMV-TCII-OLEO, pCMV-OLEO-TCII, pCMV-TCII, pCMV-OLEO, empty plasmid, pCMV-GFP-TCII-OLEO, respectively. C: Vitamin B12 binding capacity in transfected cells. ⁵⁷Co-labeled Cobalamin (Cbl, ∼300 µCi per µg) was incorporated into culture medium (30,000 dpm/mL) for three days. The total amount of radioactivity taken by each cell lines was measured in pellets and supernatants. Mean and S.E.M. are indicated. D: Indirect immunofluorescence of TCII in N1E-115 cells transiently transfected with lipofectamine. The four constructs and the empty plasmid were tranfected in N1E-115 cells (1–5). The immunofluorescence was done with a goat polyclonal antibody to TCII and a donkey antigoat IgG fluorescein labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 10 µm. E: Confocal analysis showing co-localization of the protein GFP-TCII-OLEO with endoplasmic reticulum in transfected N1E-115 cells. The cells were transfected with the plasmid pCMV-GFP-TCII-OLEO coding for GFP-transcobalamin-oleosin (GFP-TCII-OLEO), using lipofectamine. Cell nuclei were counterstained with Hoechst 33258 (1, 5). Co-localization was evidenced with fluorescence from GFP (2, 6), immuno-fluorescence with a mouse monoclonal antibody to the human golgin-97 (3) or a rabbit polyclonal antibody to calreticulin (7) and merge fluorescence (4,8). The secondary antibodies include a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Calibration bars = 20 µm.</p

Viability and apoptosis of N1E-115 cells stably transfected with different plasmids.

<p>The plasmids were pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII), pCMV-TCII coding for transcobalamin II (TCII), pCMV-OLEO coding for oleosin (OLEO), and pCDNA3. A: Cell viability and apoptosis among growth delay in proliferated state was monitored by the absorbance of formazan dye resulting from enzymatic metabolism of MTT by the mitochondrial dehydrogenase. The no reagent blank was MTT alone (0.5 mg/mL in culture medium). Western blot analysis of the temporal course of p53 in cells transfected with pCMV-TCII-OLEO was made by using a mouse anti-p53 antibody and a mouse anti-actin antibody, which were revealed with a goat anti-mouse IgG coupled to peroxidase. Cleaved Caspase-3 was identified by a rabbit anti-caspase-3 polyclonal antibody and a donkey anti-rabbit secondary antibody (labeled with peroxidase). B: Western blotting of cleaved Caspase-3 at Day 7 of proliferate state. The mean±S.E.M. were obtained from three independent experiments made in triplicate. Two-way ANOVA and Bonferoni post-test analyzed statistical differences from the control groups. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Apoptosis of tyrosine hydroxylase (TH) immunoreactive cells in the <i>substantia nigra</i> of rats transfected with several plasmids.

<p>A: TH-immunoreactive neurons after transfection. The neurons were transfected with NTS-polyplex with one of the following plasmids, pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO, 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII, 2), pCMV-TCII coding for transcobalamin II (TCII, 3), pCMV-OLEO coding for oleosin (OLEO, 4), and the pCDNA3, the empty plasmid (5). Mesencephalon slices (40 µm) were immunostained at 2-month after transfection with a mouse monoclonal antibody to TH and a donkey antimouse IgG fluorescein labeled. Representative micrographs of sagital section of the rat mesencephalon are presented. Calibration bars = 200 µm. B: Apoptosis in TH-immunoreactive neurons after transfection with the plasmid pCMV-TCII-OLEO. Representative micrographs of the <i>substantia nigra</i> (with double immunostaining at 15-day after transfection) are presented. The primary antibodies were a mouse monoclonal antibody to TH, and a rabbit polyclonal antibody to cleaved Caspase-3. The secondary antibodies included a donkey anti-mouse IgG FITC labeled (1 and 4), and a donkey anti-rabbit IgG rhodamine labeled (2 and 5). Representative micrographs of coronal section of control <i>substantia nigra</i> (1–3) and transfected <i>substantia nigra</i> (4–6) of the same rat are presented. Scale bars = 50 µm. C: Apoptosis in TH immunoreactive neurons expressing the TCII-OLEO chimera. Representative micrographs of the <i>substantia nigra</i> with triple immunostaining at 15-day after transfection. The primary antibodies were a mouse monoclonal antibody to TH, a goat polyclonal antibody to TCII, and a rabbit polyclonal antibody to cleaved Caspase-3. The secondary antibodies were a donkey anti-mouse IgG AMCA labeled (1 and 5), a donkey antigoat IgG fluorescein labeled (2 and 6), a donkey anti-rabbit IgG rhodamine labeled (3 and 7). Representative micrographs of coronal section of control <i>substantia nigra</i> (1–4) and transfected <i>substantia nigra</i> (5–8) of the same rat are presented. Scale bars = 50 µm.</p

Analysis of apoptosis in N1E-115 cells stably transfected with different plasmids.

<p>The plasmids were pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII), pCMV-TCII coding for transcobalamin II (TCII), pCMV-OLEO coding for oleosin (OLEO), and pCDNA3. The immunofluorescence was done with a rabbit polyclonal antibody to cleaved Caspase-3 and a donkey antirabbit IgG fluorescein labeled. Before fixation, cells were incubated with 4 µM propidium iodide for 10 min. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 100 µm.</p

Expression of transcobalamin II/oleosin (TCII/OLEO) chimeric proteins in rats 60 days after transfection with the NTS-polyplex.

<p>A: RT-PCR from the plasmid transcripts in the <i>substantia nigra</i> of rats. A group of rats (n = 3) was transfected with the plasmid pCMV-TCII-OLEO and another (n = 3) with the plasmid pCMV-OLEO-TCII. RT-PCR amplified a fragment of 380 bp for TCII-OLEO, a fragment of 394 for OLEO-TCII, and a fragment of 349 for β-actin, the internal control. Lane 1 corresponds to the amplified fragment from the plasmid (positive control). Lane 2 is a PCR in the absence of plasmid or cDNA (negative control). The amplified product from the transfected substantia nigra of each rat corresponds to the lanes 3, 5, and 7, and the lanes 4, 6, and 8 show the RT-PCR outcome from the non-transfected side. B: GFP immunofluorescence in the rat <i>substantia nigra</i> transfected with pCMV-GFP-TCII-OLEO. The pCMV-GFP-TCII-OLEO encodes for the fusion protein green fluorescent protein-transcobalamin-oleosin (GFP-TCII-OLEO). The immunofluorescence was done with a mouse monoclonal antibody to GFP and a donkey antimouse IgG fluorescein labeled. Representative micrographs of coronal section of control substantia nigra (1) and transfected substantia nigra (2) of the same rat are presented. Calibration bars = 100 µm. C: Double immunofluorescence against TCII and tyrosine hydroxylase (TH) in the <i>substantia nigra</i> of rats. The neurons were transfected with NTS-polyplex with pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO). Slices from mesencephalon (40 µm) were immunostained at 7-day after transfection. The primary antibodies were a goat polyclonal anti-TCII and a mouse monoclonal anti-TH. The secondary antibodies were a donkey antigoat IgG fluorescein labeled and a donkey antimouse IgG rhodamine labeled. Representative micrographs of coronal section of control <i>substantia nigra</i> (1–3) and transfected <i>substantia nigra</i> (4–6) of the same rat are presented. Calibration bars = 50 µm.</p