Reason for doing the research
The goal of the paper was to investigate whether pharmacological treatment with the pan- Peroxisome Proliferator-Activated Receptor (PPAR) agonist bezafibrate would correct a deficiency of PPAR--coactivator 1α (PGC-1 α) and apply beneficial effects in a transgenic mouse having Huntington’s disease (HD). A number of mice with a deficiency of PGC-1 α were fed on a special diet comprising of bezafibrate and another group was left untreated. The mice were examined for a long period and then euthanized with CO2 inhalation. They died after some time and were dissected with several internal parts (brain, muscles, liver etc) being examined.
The hypotheses was based on the evidence that there is substantial evidence that the impairment of PPAR-y-Coactivator 1α (PGC-1 α) levels and activity play an important role in Huntington’s Disease (HD) (Johri et al, 2011). As such, the hypotheses stated that since HD, transgenic mice show reduced levels of PGC-1 α and its downstream target genes, and then the bezafibrate treatment was expected to increase the PGC-1 α expression and mitochondrial biogenesis, which would result in improved survival, improved phenotype, and reduced muscle, brain, and brown adipose tissue (BAT) pathology in HD mice.
Methods used to carry out the experiments
This experiment was laboratory method where all conditions are fully controlled. The experiment used R6/2 mice with an N-terminal genomic fragment that contained exon1 with 130 Cytosine-Adenine-Guanine (CAG) repeats. The R6/2 mice were also raised on a diet containing 0.5% bezafibrate or standard chow after they had been weaned. This shows that the conditions of the experiment were fully controlled in order to enable the researchers to conduct the tests necessary for testing the laid out hypotheses.
The experiment also used the natural or observational method. This method entails observations on nature or natural occurrences. The researcher simply observes without controlling or altering the observable traits. The researchers observed the wild littermates of the R6/2 mice, which had not been fed with special diet. The researchers tested the brain, muscles, and BAT of the mice as they compared the results with those from mice placed under special diet. For instance, the test for motor strength involved the observation of untreated and treated mice for their movement and measuring the distance covered.
The quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the N-truncated and the full-length (FL) isoforms of the PGC-1 α are downregulated in R6/2 mice muscle, brain, and BAT compared with their wild-type mice. The N-truncated and the full-length (FL) isoforms were notably induced in the peripheral and brain tissues of R6/2 mice that on the bezaﬁbrate diet and their levels were not signiﬁcantly different from those of the wild-type controls.
The mRNA expression levels of the three isoforms of PPARs (a, g and d) and that of Cytochrome c (Cyt c), ATP and Tfam synthase recorded lower levels of the R6/2 muscle, brain, and BAT when compared with the wild-type mice of the same age. The levels of NRF-1 did not change in the BAT and the brain.
In R6/2 brains, the researchers found that genes responsive to ROS, such as nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and Gpx-1 as well as hemoxygenase-1 (HO-1) were signiﬁcantly downregulated. The presence of bezaﬁbrate in the diet of the mice restored the levels of those genes to control levels in the R6/2 mice.
When the mice were tested for muscle strength, the R6/2 mice showed robust deterioration of their grip strength as they aged as compared with their wild littermates. The differences were significant at 8, 10, and 12 weeks of age. The mice that were fed on Bezafibrate showed increased grip strength (up to 2-fold) as compared with the untreated mice.
In order to test for motor strength, the mice were tested in an open field. The R6/2 mice were significantly hypoactive when the distances they covered were measured. The Bezafibrate-treated mice showed significant hyperactivity was noted. The treated mice moved faster and longer distances as compared to the untreated mice.
The mice were also observed for striatum. The R6/2 mice that were treated with bezafibrate showed enlarged extracellular spaces, lysosome-like dense bodies, and cytoplasmic valcuoles. In the untreated mice, these traits were much subdued. Degenerated or degenerating mitochondria could also be observed at a significant rate in the treated mice as compared to those in the untreated ones. In the striata from the bezafibrate-treated R6/2 mice, the cytoplasm of the neurons is maintained and the dendritic profiles and the axonal in the neuropil are intact.
Interpretations of findings
There are several bioenergetic and metabolic impairments are known to occur in HD patients. Mice that are deficient in PGC-1 α exhibit relations of transcriptional co-activator PGC-1 α to HD pathogenesis. The PCG-1 exhibit impaired mitochondrial function, hyperkinetic movement disorder and striatal degeneration. Since the mitochondria is the cell component responsible for converting nutrients into energy, any condition that affects its functioning negatively reduces its capability to provide energy to the body and hence the lethargy in the mice that had HD. The impairment of the PGC-1 pathway can be reversed in the brain, muscle and the BAT from R6/2 in mice using the PPAR-panagonist, bezafibrate. This PPAR has been shown to be effective in increasing the lifespan and in delaying the onset of symptoms of HD in a mouse model of mitochondrial myopathy. The PPAR α also cause weight loss and hepatomegaly. The PPAR increases genes involved in fatty acid oxidation but it does not increase the mtDNA content and the mitochondrial respiratory chain activities in mice. Bezafibrate administration restored the mRNA levels of PGC-1 α and PPARs, downstream targets of PGC-1 α Cyt c and Tfam and ATP synthase in brain, muscle, and BAT of R6/2 mice. Bezafibrate improves the phenotype and the survival of R6/2 mice in a comparable range to the best therapeutic interventions that have been tested this far. The improved phenotype increased the survival and the induction of the PGC-1 signaling pathway, which are accompanied by reduced neuropathological feature. There is also a significant increase in mitochondrial density in stratum of R6/2 mice that were treated with bezafibrate. It is, therefore, true that PGC-1 a plays a critical role in mitochondrial biogenesis.
Conclusion on the findings
The experiment was successful and it managed to prove the hypotheses right. The function of the PGC-1 α in the regulation of the mitochondrial function as well as the association of mitochondrial dysfunction with HD pathogenesis was proved. This implies that activation of PGC-1a may be useful in the treatment of HD. In this experiment, the researchers proved that we show that stimulation of PPAR-PGC-1a axis by bezaﬁbrate produces widespread beneﬁcial effects in brain and peripheral tissues of R6/2 model of HD.
Johri et al (2011) Pharmacologic activation of mitochondrial biogenesis exerts widespread beneﬁcial effects in a transgenic mouse model of Huntington’s disease. Human Molecular Genetics, 2012, Vol. 21, No. 5 1124–1137 doi:10.1093/hmg/ddr541Advance Access published on November 17, 2011. Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York-Presbyterian