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

Abstract

BackgroundVitamin B12 is indispensable for proper brain functioning and cytosolic synthesis of S-adenosylmethionine. Whether its deficiency produces effects on viability and apoptosis of neurons remains unknown. There is a particular interest in investigating these effects in Parkinson disease where Levodopa treatment is known to increase the consumption of S-adenosylmethionine. To cause deprivation of vitamin B12, we have recently developed a cell model that produces decreased synthesis of S-adenosylmethionine by anchoring transcobalamin (TCII) to the reticulum through its fusion with Oleosin (OLEO).MethodologyGene constructs including transcobalamin-oleosin (TCII-OLEO) and control constructs, green fluorescent protein-transcobalamin-oleosin (GFP-TCII-OLEO), oleosin-transcobalamin (OLEO-TCII), TCII and OLEO were used for expression in N1E-115 cells (mouse neuroblastoma) and in substantia nigra of adult rats, using a targeted transfection with a Neurotensin polyplex system. We studied the viability and the apoptosis in the transfected cells and targeted tissue. The turning behavior was evaluated in the rats transfected with the different plasmids.Principal FindingsThe transfection of N1E-115 cells by the TCII-OLEO-expressing plasmid significantly affected cell viability and increased immunoreactivity of cleaved Caspase-3. No change in propidium iodide uptake (used as a necrosis marker) was observed. The transfected rats lost neurons immunoreactive to tyrosine hydroxylase. The expression of TCII-OLEO was observed in cells immunoreactive to tyrosine hydroxylase of the substantia nigra, with a superimposed expression of cleaved Caspase-3. These cellular and tissular effects were not observed with the control plasmids. Rats transfected with TCII-OLEO expressing plasmid presented with a significantly higher number of turns, compared with those transfected with the other plasmids.Conclusions/SignificanceIn conclusion, the TCII-OLEO transfection was responsible for apoptosis in N1E-115 cells and rat substantia nigra and for Parkinson-like phenotype. This suggests evaluating whether vitamin B12 deficit could aggravate the PD in patients under Levodopa therapy by impairing S-adenosylmethionine synthesis in substantia nigra.

Cobalamin and normal prions: A new horizon for cobalamin neurotrophism

“…No histological signs of apoptosis were observed in any SC region of Cbl-D rats, despite reports of apoptosis have been reported by others in the CNS of TNF-a-treated rats [24,25], in cultured Cbl-deprived neoplastic cells [26], and, more recently, in cultured neuroblastoma cells transfected with plasmid containing transcobalamin-oleosin, a very efficient chelator of Cbl [27].…”

Luminal expression of cubilin is impaired in Imerslund-Grasbeck syndrome with compound AMN mutations in intron 3 and exon 7

“…15,16 IF-Cbl receptor activity was assessed in cell culture medium by gel filtration chromatography, as previously described. 4 …”

Integral Proteins in Plant Oil Bodies

“…Several biotechnological applications have been developed by using the unique characteristics of oil bodies, such as new ingredients for flavoring or emulsifying agents, affinity matrices for enzyme fixation/purification, and expression/purification systems for producing recombinant proteins via transgenic plants [144][145][146][147][148][149][150][151][152][153][154]. Many applications related to the utilization of oil bodies have been patented [155][156][157][158][159][160][161][162][163].…”