The m.3243A>G mtDNA mutation is pathogenic in an in vitro model of the human blood brain barrier

Abstract

MELAS is a common mitochondrial disease frequently associated with the m.3243A>G point mutation in the tRNA(Leu(UUR)) of mitochondrial DNA and characterized by stroke-like episodes with vasogenic edema and lactic acidosis. The pathogenic mechanism of stroke and brain edema is not known. Alterations in the blood brain barrier (BBB) caused by respiratory chain defects in the cortical microvessels could explain the pathogenesis. To test this hypothesis we developed a tissue culture model of the human BBB. The MELAS mutation was introduced into immortalized brain capillary endothelial cells and astrocytes. Respiratory chain activity and transendothelial electrical resistance, TEER was measured. Severe defects of respiratory chain complex I and IV activities, and a moderate deficiency of complex II activity in cells harboring the MELAS mutation were associated with low TEER, indicating that the integrity of the BBB was compromised. These data support our hypothesis that respiratory chain defects in the components of the BBB cause changes in permeability.

Fig.1

Repopulation of endothelial cells and astrocytes with the m.3243A>G mtDNA mutation: scheme.

Fig.2

RFLP analysis of the MELAS mutation. (A) WT and MELAS endothelial cells and astrocytes. 1:Size marker, 2:Uncut, 3:MELAS cybrids, RN164, 4:hCMEC/D3-Clone 2 , 5:hCMEC/D3-Clone 10, 6:hCMEC/D3 WT 1, 7: hCMEC/D3 WT 2, 8:IHFA Clone 16, 9:IHFA Clone 21, 10: MELAS cybrids 11: IHFA WT and (B) WT and MELAS primary astrocytes. 1:MAG1, MELAS primary astrocytes, 2:WT primary astrocytes. WT cells show the 192 kb band; Mutant cells reveal the 97 and 72 kb diagnostic bands.

Fig.3

Quantitation of mtDNA/nDNA ratios in the WT and MELAS EC and astrocyte clones. WT EC, (hCMEC/D3), MELAS EC clones, (hCMEC/D3-2 and hCMEC/D3-10), WT astrocytes (IHFA) and MELAS astrocyte clones, (IHFA-16 and IHFA-21). Outset: There is no significant change in the mtDNA/nDNA ratio in WT and MELAS cells during repopulation.

Fig.4

COX and SDH histochemistry of WT and MELAS EC and astrocytes. (A), WT and MELAS EC clones, (hCMEC/D3-2 and hCMEC/D3-10), and (B), WT and MELAS astrocyte clones, (IHFA-16 and IHFA-21). Staining for COX was reduced in the MELAS EC and astrocyte clones; SDH staining was reduced in both the MELAS EC clones and in one MELAS astrocyte clone.

Fig.5

Mitochondrial translation and function in primary MELAS and WT astrocytes. Immunostaining for mitochondrial COX subunit II is reduced in primary MELAS astrocytes, MAG1 (a) compared to normal (b), while the staining for nuclear encoded subunit PDH is normal in both (c, d) indicating defective translation of mitochondrial subunits. Histochemical staining for COX is low in the MELAS astrocytes (e) compared to normal (f); SDH histochemistry is normal in both (g, h).

Fig.6

TEER of WT (hCMEC/D3) and MELAS endothelial cell clones. Mutant clones, (hCMEC/D3 Cl 10 and 2) had lower TEER readings compared to WT (hCMEC/D3) at every time point analyzed indicating increased permeability.