第665回 千葉医学会例会・第18回 佐藤外科例会 10.

Metabolic dysfunction is well-documented in Huntington's disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD

Comparison of mRNA in healthy and HD lymphoblasts and their responses to serum deprivation and cyanide treatment (95% confidence interval).

<p>All changes are significant (p≤0.05) except for in the third column of panel C.</p

Genes associated with the enzymes of the TCA cycle, PDHC and transketolase.

<p>Genes associated with the enzymes of the TCA cycle, PDHC and transketolase.</p

HD cells respond uniquely to metabolic stress.

<p>(A) Growth rates were established and compared during the initial stages of the study. Cells were always seeded at 2x10⁵ cells/ml and left to grow for 3.5 days before passaging. Two healthy and two HD cell lines were counted 14 times each (n = 28) using a hemocytometer. Live cell counts were approximately equal between healthy (1.32x10⁶) and HD (1.43x10⁶) lines after 3.5 days of growth. (B) Lymphoblasts were seeded as described in the ‘Tissue culture maintenance’ section in serum-free media. The total number of live cells was monitored every 24 hours for 72 hours (n = 3). The error bars represent standard errors of the mean. (C) Death of lymphoblasts under serum, serum-free, and serum-free plus cyanide conditions. Cell death was assessed as described in the ‘Live/dead analysis’ section. Cell death was measured in triplicate per flask and then averaged. Each experiment consisted of three independent flasks. The average cell death of the three flasks together was determined to be the percent death for that specific cell line for that particular experiment (n = 1). The treatment was repeated 3–4 more times per cell line. Thus, the cell death for each cell line for each treatment was determined to be the average of 3–4 experiments (essentially triplicate or quadruplicate experiments of identical triplicate flasks, each of which was measured in triplicate). This process was repeated for all 16 cell lines (n = 8 healthy, n = 8 HD). Values are the means ± SEM. The average death rates for each individual cell line is plotted to show consistency of trends. (D) The average change in cell death induced by removal of serum for 54 hours and additional death induced by addition of 1 mM NaCN for 6 hours after 48 hours of serum-deprivation. The average cell death for each cell line was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160384#pone.0160384.g002" target="_blank">Fig 2A</a>. The difference in cell death between serum and serum-free conditions was calculated based on these averages for each cell line. The deltas in death for all 8 cell lines were then averaged to determine the final average change in cell death induced by removing serum from the media for each subject group (healthy and HD). The same process was repeated for the cyanide treatment. *p≤0.05 determined by Student t-test. **p≤0.01 determined by Student t-test.</p

Western blot analysis.

<p>(A) A typical MDH Western blot is shown. Four arbitrarily chosen healthy and four HD lines were measured using 7.5 μg of lymphoblast lysate per well. MDH protein was reduced in the HD lines by 47% (p≤0.05). (B) A typical PDHC Western blot is shown. Three arbitrarily chosen healthy and three HD lines were measured using 15 μg of lymphoblast lysate per well. The Western blot was repeated with three different sets of cell lines with similar results (not shown). (C) A typical ICDH Western blot is shown. Seven healthy and seven HD lines were measured using 7.5 μg of lymphoblast lysate per well (4 cell lines for each sample group are shown). (D) Quantification of the measured protein levels is provided. The intensities of the β-actin bands were normalized to the highest intensity. Then the intensities of the specified protein bands were divided by the normalized intensities of the β-actin bands for each respective measurement. Differences were assessed by comparing the ratio of the specified protein to normalized β-actin for each cell line. The MDH and ICDH blots were repeated using the same cell lines (not shown). The final ratios for each cell line and the repeated measurements were averaged and compared using a Student’s t-test. Error bars represent SEM. * p≤0.05 determined by Student t-test.</p

Patient criteria for selecting lymphoblast cultures.

<p>Patient criteria for selecting lymphoblast cultures.</p