Effect of post-mortem delay on N-terminal huntingtin protein fragments in human control and Huntington disease brain lysates.

Huntington disease is associated with elongation of a CAG repeat in the HTT 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.

Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full-length HTT or an N-terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP-positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50-61.

Making (anti-) sense out of huntingtin levels in Huntington disease.

BACKGROUND: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the first exon of the HTT gene, resulting in an expanded polyglutamine tract at the N-terminus of the huntingtin protein. Typical disease onset is around mid-life (adult-onset HD) whereas onset below 21 years is classified as juvenile HD. While much research has been done on the underlying HD disease mechanisms, little is known about regulation and expression levels of huntingtin RNA and protein. RESULTS: In this study we used 15 human post-mortem HD brain samples to investigate the expression of wild-type and mutant huntingtin mRNA and protein. In adult-onset HD brain samples, there was a small but significantly lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while wild-type and mutant huntingtin protein expression levels did not differ significantly. Juvenile HD subjects did show a lower expression of mutant huntingtin protein compared to wild-type huntingtin protein. Our results in HD brain and fibroblasts suggest that protein aggregation does not affect levels of huntingtin RNA and protein. Additionally, we did not find any evidence for a reduced expression of huntingtin antisense in fibroblasts derived from a homozygous HD patient. CONCLUSIONS: We found small differences in allelic huntingtin mRNA levels in adult-onset HD brain, with significantly lower mutant huntingtin mRNA levels. Wild-type and mutant huntingtin protein were not significantly different in adult-onset HD brain samples. Conversely, in juvenile HD brain samples mutant huntingtin protein levels were lower compared with wild-type huntingtin, showing subtle differences between juvenile HD and adult-onset HD. Since most HD model systems harbor juvenile repeat expansions, our results suggest caution with the interpretation of huntingtin mRNA and protein studies using HD cell and animal models with such long repeats. Furthermore, our huntingtin antisense results in homozygous HD cells do not support reduced huntingtin antisense expression due to an expanded CAG repeat.

Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

Huntington disease is caused by expansion of a CAG repeat in the huntingtin gene that is translated into an elongated polyglutamine stretch within the N-terminal domain of the huntingtin protein. The mutation is thought to introduce a gain-of-toxic function in the mutant huntingtin protein, and blocking this toxicity by antibody binding could alleviate Huntington disease pathology. Llama single domain antibodies (VHH) directed against mutant huntingtin are interesting candidates as therapeutic agents or research tools in Huntington disease because of their small size, high thermostability, low cost of production, possibility of intracellular expression, and potency of blood-brain barrier passage. We have selected VHH from llama phage display libraries that specifically target the N-terminal domain of the huntingtin protein. Our VHH are capable of binding wild-type and mutant human huntingtin under native and denatured conditions and can be used in Huntington disease studies as a novel antibody that is easy to produce and manipulate.

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection.

BACKGROUND: Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge. RESULTS: HRMA results nicely matched those obtained with ELISA and compared favourably to DNA fingerprinting of restriction digested clone insert-PCR. DNA sequence analysis confirmed that HRMA-clustered clones contained identical inserts. CONCLUSION: Using HRMA, analysis of up to 384 samples can be done simultaneously and will take approximately 30 minutes. Clustering of clones can be largely automated using the system's software within 2 hours. Applied to the analysis of clones obtained after phage display antibody selection, HRMA facilitated a quick overview of the overall success as well as the identification of identical clones. Our approach can be used to characterize any clone set prior to sequencing, thereby reducing sequencing costs significantly.