Abstract
Dysfunctions of Ca2+ homeostasis and of mitochondria have been studied in immortalized striatal cells from a commonly used Huntington disease mouse model. Transcriptional changes in the components of the phosphatidylinositol cycle and in the receptors for myo-inositol trisphosphate-linked agonists have been found in the cells and in the striatum of the parent Huntington disease mouse. The overall result of the changes is to delay myo-inositol trisphosphate production and to decrease basal Ca2+ in mutant cells. When tested directly, mitochondria in mutant cells behave nearly normally, but are unable to handle large Ca2+ loads. This appears to be due to the increased Ca2+ sensitivity of the permeability transition pore, which dissipates the membrane potential, prompting the release of accumulated Ca2+. Harmful reactive oxygen species, which are produced by defective mitochondria and may in turn stress them, increase in mutant cells, particularly if the damage to mitochondria is artificially exacerbated, for instance with complex II inhibitors. Mitochondria in mutant cells are thus peculiarly vulnerable to stresses induced by Ca2+ and reactive oxygen species. The observed decrease of cell Ca2+ could be a compensatory attempt to prevent the Ca2+ stress that would irreversibly damage mitochondria and eventually lead to cell death.
Highlights
Huntington disease (HD)2 is a fatal disease characterized by chorea and psychiatric disturbance (1) caused by the expansion of CAG repeats in the first exon of the gene encoding huntingtin (Htt)
Calcium Signaling and Mutated Huntingtin pensity of the permeability transition pore to open: recent findings have shown that the membrane potential (⌬m) in mitochondria of cells expressing mutant huntingtin is peculiarly sensitive to Ca2ϩ (17), and that poly-Q constructs de-energize isolated mitochondria exposed to Ca2ϩ (17)
The transcription of Reactive oxygen species (ROS)-scavenging enzymes is down-regulated, and the concentration of ROS in the neurons increases. These important observations were reinforced by the finding that striatal neurons from HD knock-in mice were resistant to 3-nitropropionic acid (3-NPA) when expressing exogenous PGC-1␣ (22)
Summary
Clonal striatal cell lines established from E14 striatal primordia of KI-HdhQ111 and WT-HdhQ7 littermate mouse embryos were described previously (23). The cells were grown in Dulbecco’s modified essential medium (EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (EuroClone), 2 mM glutamine, 10 units/ml penicillin, 100 g/ml streptomycin. They were maintained at the permissive temperature (33 °C) in a humidified incubator with 5% CO2. Stock solutions in water of ATP (100 mM) and BK (1 mM) (Sigma, St. Louis, MO) were prepared, aliquoted, and stored at Ϫ20 °C. The luminescence data were converted off-line into [Ca2ϩ] values, using a previously described computer algorithm (25). Fura-2 images were collected every 3 s and the 340/380 ratio values were normalized as described above
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