Several lines of evidence have recently suggested that abnormal Ca2+ uptake capacity is involved in HD neurons.
Oliveira and Goncalves investigated the buffering capacity of mitochondrial Ca2+
in cortical and striatal neuron-astrocyte co-cultures (Oliveira and Goncalves, 2009
). They found that mitochondria not only in neurons but also in astrocytes from striatal origin exhibited a decrease in mitochondrial Ca2+
buffering capacity when compared with cortical counterparts. The decrease in this buffering capacity did not stem from variations in mitochondrial concentration or in the rate of intracellular Ca2+
elevation, but was mechanistically linked to an increased propensity of the mitochondria to undergo cyclosporin A-sensitive permeability transition. Indeed, 1 μM cyclosporin A selectively was found to increase the mitochondrial Ca2+
buffering capacity of striatal astrocytes, without modifying the neurons or cortical astrocytes. Neither the thapsigargin nor FK506 modified mitochondrial Ca2+
buffering in between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provided additional evidence into the mechanisms of striatal vulnerability, showing an increase in Ca2+
vulnerability of striatal versus cortical mitochondria, in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions (Oliveira and Goncalves, 2009
Rockabrand et al studied the mutant Htt subcellular localization, aggregation and intracellular Ca2+ dynamics in PC12 cells expressing various domains of mutant Htt (Rochabrand et al., 2007). They found that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Htt exon1 region mitochondrial localization and also promoting association with the endoplasmic reticulum and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyglutamine repeats acutely disrupts intracellular Ca2+ levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Htt exon 1region resulted in uncoupling and depolarization of these organelles, further supporting the idea that Htt exon1-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis (Rochabrand et al., 2007).
Milakovic et al. elucidated the effects of Ca2+
on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) Htt-expressing cells of striatal origin (Milakovic et al., 2006
). When treated with increasing Ca2+
concentrations, mitochondria from mutant Htt-expressing cells showed increased sensitivity to Ca2+
, since mitochondria from mutant Htt-expressing cells were more sensitive to Ca2+
-induced decreases in state 3 respiration and DeltaPsim, than were mitochondria from wild-type cells. Further, mutant Htt-expressing cells had a reduced mitochondrial Ca2+
uptake capacity in comparison with the capacity of wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The DeltaPsim defect was attenuated in the presence of ADP, and the decreases in Ca2+
uptake capacity were abolished in the presence of mitochondrial permeability transition pore inhibitors. These findings indicate that mutant Htt-expressing cells have mitochondrial Ca2+
handling defects that result in respiratory deficits and that the increased sensitivity of mutant Htt to Ca2+
induced mitochondrial permeabilization may be a contributing mechanism to mitochondrial dysfunction in HD.
Lim et al. investigated dysfunctions of Ca2+
homeostasis in mitochondria, in striatal neurons from postmortem brains of HD patients (Lim et al., 2008
). They found mitochondria in mutant striatal neurons behaved normally, but are unable to handle large Ca2+
loads, may due to the increased sensitivity of Ca2+
to the permeability transition pore opening, which dissipates the membrane potential, prompting the release of accumulated Ca2+
. Harmful ROS, produced by defective mitochondria and possibly stressing them, increases in mutant cells, particularly if the damage to mitochondria is artificially exacerbated with, for example, complex II inhibitors. Mitochondria in mutant cells are thus peculiarly vulnerable to stresses induced by Ca2+
and ROS. The observed decrease of cell Ca2+
could be a compensatory attempt to prevent Ca2+
stress that would irreversibly damage mitochondria and eventually lead to cell death.
Gellerich et al. studied brain mitochondria of transgenic HD rats with 51 glutamine repeats, which modeled the adult form of HD (Gellerich et al. 2008
). Ca(free)(2+) up to 2 μM activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition. Ruthenium red, an inhibitor of the mitochondrial Ca2+
uniporter, did not affect the Ca2+
-dependent activation of respiration but reduced the Ca2+
-induced inhibition. Thus, Ca2+
activation was mediated exclusively by extramitochondrial Ca2+
, whereas Ca2+
inhibition was promoted by intramitochondrial Ca2+
. In contrast, Htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+
activation, and a higher susceptibility to Ca2+
dependent inhibition. Htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca2+
, lower capacities and rates of Ca2+
accumulation, and a decreased Ca2+
threshold for permeability transition in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+
induced inhibition of respiration of Htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extra-mitochondrial Ca2+
. This study concluded that interactions between Htt(51Q) and distinct targets such as aralar and/or the permeability transition pore may underlie mitochondrial dysregulation, leading to energetic depression, cell death, and tissue atrophy in HD.
Fernandes et al. investigated the connection between NMDA receptors and Ca2+
in full-length YAC HD transgenic mice expressing 128 polyglutamine repeats (Fernandes et al., 2007
). NMDA-induced apoptosis were found to be enhanced in YAC128 medium spiny neurons in this mouse model. However, initial steps in the death-signaling pathway, including NMDA receptor current and cytosolic Ca2+
loading, were similar to those observed in wild-type medium spiny neurons. They also found that the NMDA receptor -mediated Ca2+
load triggered a strikingly enhanced loss of mitochondrial membrane potential in YAC128 medium spiny neurons, suggesting that NMDAR signaling via the mitochondrial apoptotic pathway is altered. This effect was accompanied by impaired cytosolic Ca2+
clearance after removal of NMDA, a difference that was not apparent after high potassium-evoked depolarization-mediated Ca2+
entry. Inhibition of the mitochondrial permeability transition reduced peak cytosolic Ca2+
and mitochondrial depolarization evoked by NMDA in YAC128 medium spiny neurons but not wild-type medium spiny neurons. These results suggest that the polyglutamine repeat length influences the mechanism by which mutant Htt enhances NMDA receptor-mediated excitotoxicity (Fernandes et al., 2007
Oliveira et al. investigated bioenergetic behavior of mitochondria isolated from the from fore brains of R6/2 mice, YAC128 mice, and Hdh150 knock-in mice and wild-type littermates using in situ
respiratory parameters in intact HD striatal neurons (Oliveira et al., 2007
). They assessed the Ca2+
loading capacity of isolated mitochondria by steadily infusing Ca2+
. Mitochondria from 12–13 weeks old R6/2 mice and 12 months old YAC128 mice, but not homozygous or heterozygous Hdh150 knock-in mice (15–17 weeks), exhibit increased Ca2+
loading capacity when compared to non-transgenic, control mice. In situ
mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full-length mutant Htt does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy-demanding stimuli, Hdh150 neurons are more vulnerable to Ca2+
deregulation than neurons from nontransgenic, wild-type mice. These findings suggest to assess the HD mitochondrial function in the cellular context(Oliveira et al., 2007
Using HD mouse models and real-time functional imaging of intracellular Ca2+
and mitochondrial membrane potential, Oliveira and colleagues studied the relationship between mitochondria and Ca2+
handling in intact HD striatal neurons (Oliveira et al., 2006
). They treated HD striatal neurons with histone deacetylase inhibitors, which are known to protect neurons. This treatment reduced cell death in the HD models, but its effects on cellular function are unknown. Using use real-time functional imaging of intracellular Ca2+
and mitochondrial membrane potential, they explored the role of in situ HD mitochondria in Ca2+
handling. Immortalized striatal cells and striatal neurons from transgenic mice expressing full-length mutant Htt were used to model HD. They found that active glycolysis in HD striatal neurons occludes the mitochondrial role in Ca2+
handling as well as the effects of mitochondrial inhibitors, HD striatal neurons and striatal neurons in the absence of glycolysis are critically dependent on oxidative phosphorylation for energy-dependent Ca2+
handling, expression of full-length mutant Htt is associated with deficits in mitochondrial-dependent Ca2+
handling that can be ameliorated by treatment with histone deacetylase inhibitors, and neurons with different response patterns to NMDA receptor activation exhibit different average somatic areas and are differentially affected by treatment with histone deacetylase inhibitors, suggesting subpopulation or functional state specificity. These findings indicate that neuroprotection induced by histone deacetylase inhibitors involves more efficient Ca2+
handling, thus improving the neuronal survival.
A closer examination of in vitro and in vivo studies of Ca2+ influx, mutant Htt and mitochondria reveal the following: 1) when treated with increasing Ca2+ concentrations, mitochondria from mutant Htt-expressing cells showed increased sensitivity to Ca2+, and decreased mitochondrial Ca2+ uptake capacity compared to wild type Htt expressing cells, 2) mutant Htt induce intracellular Ca2+ in HD neurons, 3) mutant Htt induced intracellular Ca2+ increases with polyglutamine repeat length in HD neurons, and 4) increased intracellular Ca2+ enter mitochondria and promote the opening of mitochondrial permeability transition pores, and damage HD neurons.
However, contrary to the above, in a recent study by Oliveira and colleagues found increased Ca2+ uptake capacity in forebrain mitochondria from 2 transgenic mice lines (R6/2 and YAC128) but not in HD knockin mice expressing 150 polyglutamine repeats. These authors used in situ hybridization techniques for the first time and assessed Ca2+ uptake capacity, and in situ hybridization technique is more reliable in assessing mitochondrial Ca2+ uptake capacity. Further research is needed to resolve the conflicting findings reported by Oliveira and colleagues using in situ hybridization technique by other groups.
Overall, overwhelming evidence clearly suggests that mutant Htt induce intracellular Ca2+ in neurons affected by HD and increased intracellular Ca2+ excessively enter mitochondria and induce to open the mitochondrial permeability transition pores, leading to decreased mitochondrial ATP, neuronal death, and ultimate tissue atrophy in HD brain.