7 research outputs found
Effect of post-mortem delay on N-terminal huntingtin protein fragments in human control and Huntington disease brain lysates
<div><p>Huntington disease is associated with elongation of a CAG repeat in the <i>HTT</i> gene that results in a mutant huntingtin protein. Several studies have implicated N-terminal huntingtin protein fragments in Huntington disease pathogenesis. Ideally, these fragments are studied in human brain tissue. However, the use of human brain tissue comes with certain unavoidable variables such as post mortem delay, artefacts from freeze-thaw cycles and subject-to-subject variation. Knowledge on how these variables might affect N-terminal huntingtin protein fragments in post mortem human brain is important for a proper interpretation of study results. The effect of post mortem delay on protein in human brain is known to vary depending on the protein of interest. In the present study, we have assessed the effect of post mortem delay on N-terminal huntingtin protein fragments using western blot. We mimicked post mortem delay in one individual control case and one individual Huntington disease case with low initial post mortem delay. The influence of subject-to-subject variation on N-terminal huntingtin fragments was assessed in human cortex and human striatum using two cohorts of control and Huntington disease subjects. Our results show that effects of post mortem delay on N-terminal huntingtin protein fragments are minor in our individual subjects. Additionally, one freeze-thaw cycle decreases the huntingtin western blot signal intensity in the cortex control subject, but does not introduce additional N-terminal huntingtin fragments. Our results suggest that subject-to-subject variation contributes more to variability in N-terminal huntingtin fragments than post mortem delay.</p></div
N-terminal huntingtin fragments in control temporal cortex tissue from an individual subject.
<p>Western blot analysis of temporal lobe tissue samples from tissue with no (A) and one freeze-thaw cycle (B) before sampling. Upper blot: full length htt. Middle and lower blot: N-terminal htt fragments with β-actin loading control. Squares: position of bands that increase with increasing artificial PMD (50kDa = white, 65kDa = grey). kDa = Molecular weight in kilodaltons, Artificial post mortem delay in hours (hr). (C) Whisker boxplot showing image J quantification of PMD-related htt bands at indicated timepoints relative to total htt (0 freeze-thaw cycli). (D) Whisker boxplot showing image J quantification of PMD-related htt bands at indicated timepoints relative to total htt (1 freeze-thaw cyclus). Boxes indicate the first and third quartile around the median (50 kDa = white, 65kDa = grey). Data points are shown as dots and represent four technical replicas of the same sample.</p
Comparison of N-terminal huntingtin fragments in human post mortem control and HD cortex and striatum tissue.
<p>Western blot analysis of (A) Cortex control subjects, (B) Cortex HD subjects, (C) Striatum control subjects and (D) Striatum HD subjects. Control and HD subjects were age, gender and post mortem delay matched. Upper blot: full length htt (fl htt). Middle and lower blot: N-terminal htt fragments with β-actin loading control. Arrows: position of bands associated with a post mortem delay related increase in intensity. kDa = Molecular weight in kilodaltons, Post mortem delay in hours (hr). High sensitivity: Blot analyzed at a higher viewing-sensitivity. mut = full length mutant htt, wt = full length wild-type htt.</p