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Mutant Huntingtin Gene Expression and Cell Death: A Mitochondrial Basis for Huntington�s Disease

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Title Mutant Huntingtin Gene Expression and Cell Death: A Mitochondrial Basis for Huntington�s Disease
 
Creator Chakraborty, Joy
 
Subject Cell Biology & Physiology
 
Description Cellular signalling pathways affecting normal neuronal morphology and mitochondrial quality in two different models of Huntington’s disease (HD), 3-nitropropionic acid (3-NP)-induced HD in rats and 150Q Htt expressing cell line model, were investigated for correcting the errors with the help of pharmacological agents. We used a cell line based model of HD where cells stably express N-terminal end of huntingtin (htt) upon induction, either htt with 16 CAG (taken as control) or 150 CAG (taken as the diseased condition) repeats. This model consistently showed formation of aggregates, loss in ubiquitin proteasomal system (UPS) activity, disruption of normal mitochondrial membrane potential, loss of mitochondrial electron transport chain (ETC) complex activity and cell death. Additionally, we found the imbalance in the mitochondrial dynamics in this model shifted towards the fragmentation of the organelle. For in vivo experiments, we used a chemically induced animal model of HD where the disease pathology was initiated by using 3-NP, an irreversible inhibitor of ETC complex II. This model showed impairment in gait, beam balancing, swimming ability, inability to coordinate fine motor movements and cognition. Histopathology of the brain exhibited lesion formation in the dorsolateral striatum (typical to HD), proliferation of microglia and scarcity of astroglia inside the lesion core. There was a severe loss in normal neuronal morphology with less spine formation in dendrites of the striatal and cortical neurons, mainly because of the altered interaction of profilin2 with β-actin. 3-NP treated HD rats showed increased striatal dopamine and glutamate levels, and serotonin metabolism, without affecting serotonin levels. At the mitochondrial level, a gross inhibition in ETC complex activity, decreased oxygen consumption and intensified mitochondrial fission was discernable.
Melatonin was found to correct cell viability, mitochondrial membrane potential and complex II activity in the cell line model. The mechanism was found to be mediated by the neurohormone’s ability to restore the proteasomal machinery and decreased mitochondrial fission. In the animal model melatonin was not able to protect the striatal lesion formation and the increase in striatal dopamine levels, but showed improvement in the behavioural deficiencies, which is attributed to melatonin’s protective effect on maintaining the dendritic spine density and cerebellar granule cell arborisation. N-acetyl cysteine (NAC) improved cell viability in the cellular model of HD by improving the mitochondrial quality. It was found that NAC treatment reduced the mitochondrial fission by improving the degradation of DRP1 protein. In animal model it was found that NAC administration protected striatum from lesion formation and decreased localization of DRP1 onto the mitochondria. NAC exhibited limited effect on the ubiquitin-proteasome system (UPS) activity and DA
mediated increase in the total DRP1 level. Localization of DRP1 to mitochondria and its oligomerization was found to be crucial for striatum specific neuronal loss.
Quercetin failed to improve cell viability in the cell line model of HD, and was found to be toxic when higher doses (40-100 μM) were administered. A narrow therapeutic window was seen for about 20 μM dose of quercetin, which protected the mHtt-induced cell death on 2nd day of mHtt expression. At this dose an improved UPS activity was obtained on the 4th day of induction, but did not improve mHtt mediated loss in mitochondrial membrane potential. When administered in the animal model of HD, quercetin improved animal behaviour and serotonin metabolism. It was found that quercetin was able to reduce the microglial proliferation inside the striatal lesion core with improved population of astrocytes. It has to be noted that both the models are distinctly different, the cell line model expresses mHtt which triggers the mitochondrial localization of DRP1 and thus imbalances the mitochondrial dynamics. On the other hand, we used a neurotoxin which is specific for ETC complex II inhibition and a potent oxidative stress generator. These effects lead to the increase in DRP1 and decreased Mfn 1/2 level in the animal model. Therefore, both these models together complete all the aspect of disease related mitochondrial abnormalities. Based on these results, it is concluded that mitochondrial fission control and its impact on cell death culminating into the behavioural abnormalities, maintenance of DRP1 homeostasis by UPS activity and finally the oligomerization of the DRP1 protein are the three levels at which therapeutic ventures could be attempted. In the present thesis Htt transgenic cellular model, and a chemical lesioned HD rat model were used for evaluating the therapeutic potential of three different pharmacophores. This kind of a comparative study is for the first time in literature, to the best of the author’s knowledge. In a nutshell, the outcome of the study showed NAC to reproduce all the effects in the cellular model in animal model too, whereas melatonin reproduced most of the effects in cell line model in the animal model, but quercetin failed to show complete congruence in results in cellular and animal models. These unexpected outcome of the study clearly point insufficiency of either of these models, and therefore the best combination could be a chemical lesioned animal model along with a transgenic animal model. This could be true since transgenic animal models are known to suffer from compensatory changes in the gene expression patterns of the counteracting molecules (as for eg. SOD knockout animals have upregulated catalase synthetic machinery!). The limitations of the present study being thus, the author could have adapted to a co-culture regimen (i.e., the neuronal cell line to be cultured with astrocytes to obtain a natural environment) for more meaningful results.
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2061/1/Thesis_Final_31%2D1%2D2014.pdf
Chakraborty, Joy (2014) Mutant Huntingtin Gene Expression and Cell Death: A Mitochondrial Basis for Huntington�s Disease. PhD thesis, Jadavpur University.
 
Relation http://www.eprints.iicb.res.in/2061/