By: Lise Munsie, PhD
In human studies…
Human studies provide the most useful research data and help to elucidate the natural history, biology, and potential molecular mechanisms of disease, which inform treatment development.
Dr. Jong-min Lee’s group investigated the correlation between CAG repeat length in the mutant and wildtype alleles, and age at death and disease duration.1 Similarly for age at onset of motor symptoms of HD, age at death had a strong negative correlation with mutant allele CAG repeat length. Interestingly, CAG repeat length did not correlate with disease duration, even in juvenile HD.
Nielsen and colleagues investigated the hypothesis that metabolic changes characteristic of HD may be caused in part by liver damage.2 The group used a range of common blood tests to evaluate liver function in symptomatic HD patients. Although the study has some limitations—including using data from patients on medications that may impair liver function—they found evidence of increased liver dysfunction in symptomatic HD patients. More sensitive markers of liver failure may be needed to solidly ascertain how and when the liver is affected over the course of disease.
De Souza and colleagues investigated the role of differential DNA methylation in HD, characterizing tissue specificity by examining genome-wide methylation changes in brain and liver tissue samples from HD patients.3 The study revealed DNA methylation differences in the HTT gene region between matched brain and liver samples. The study also found evidence that the transcription factor CTCF participates in DNA methylation and contributes to tissue-specific methylation patterns near the HTT proximal promoter.
1Keum JW, Shin A, Gillis T, et al. The HTT CAG-expansion mutation determines age at death but not disease duration in Huntington disease. Am J Hum Genet. 2016;98(2):287-298.
2Nielsen SM, Vinther-Jensen T, Nielsen JE, et al. Liver function in Huntington’s disease assessed by blood biochemical analyses in a clinical setting. J Neurol Sci. 2016;362:326-332.
3De Souza RA, Islam SA, McEwen LM, et al. DNA methylation profiling in human Huntington’s disease brain. Hum Mol Genet. 2016 Mar 6. pii: ddw076. [Epub ahead of print].
In animal models…
With the exception of using patient data, model organisms are the most biologically relevant method for studying disease. In an article published in Nature Neuroscience, Langfelder and colleagues used a variety of HD models—mainly murine—to perform tissue-, CAG length-, and age- dependent, large-scale, transcriptome and proteome comparisons.1 They identified a set of genes and corresponding proteins that are affected in a tissue- , CAG-, and age- dependent manner. The set includes previously identified genes such as CTCF, validating this method. All the data is freely available for viewing or data mining at www.hdinhd.org.
Although mammalian models have shed light on many aspects of HD, the classic models have yet to elucidate the neural mechanism by which striatal degeneration leads to a movement disorder. The Mooney group published their work exploring this mechanism in an unusual model—the songbird—in Proceedings of the National Academy of Sciences.2 They used lentivirus to express mHTT exon1 in the basal ganglia nucleus of a male zebra finch, and analyzed the bird’s vocal behaviors. They uncovered the affected neural pathways, and infer how this relates to movement disorders in human HD.
Although mice are easy to use in the lab, larger animal models are more indicative of human pathology. To this end, a transgenic HD sheep has been created. Though the sheep is not itself symptomatic, it can be used to study presymptomatic HD. In Nature Scientific Reports, Handley and colleagues describe their work examining metabolic profiles in different tissues from the HD sheep using gas chromotography mass spectrometry.3 They found specifically that amino acids in the cerebellum were altered, whereas fatty acids in the liver were altered. Their results suggest a hyper-metabolic defect in the sheep.
1Langfelder P, Cantle JP, Chatzopoulou D, et al. Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice. Nat Neurosci. 2016 Apr;19(4):623-633.
2Tanaka M, Singh Alvarado J, et al. Focal expression of mutant huntingtin in the songbird basal ganglia disrupts cortico-basal ganglia networks and vocal sequences. Proc Natl Acad Sci USA. 2016 Mar 22;113(12):E1720-7.
3Handley RR, Reid SJ, Patassini S, et al. Metabolic disruption identified in the Huntington’s disease transgenic sheep model. Sci. Rep. 2016 Feb 11;6:20681.
In cell models…
Huntingtin knockdown in either a pan- or allele-specific manner is one of the hottest areas of HD research. Using fibroblasts derived from HD patients and new techniques in genome editing, the Nolta group identified single nucleotide polymorphisms (SNPs) specific to the mutant allele, and were able to deliver transcription activator-like effector nucleases (TALEN) to the cells to specifically silence the mutant allele.1 The group identified a novel method for targeting and shortening the polyglutamine tract, particularly in the mutant allele.
Although knocking down the wild-type Huntingtin gene (HTT) may be therapeutic, off-target effects remain unknown. Lopes and colleagues used neural precursor cells derived from HD human embryonic stem cells to investigate the impact of mHTT on HTT’s normal function in regulating molecular motors and spindle pole orientation.2 They demonstrated that a repeat length of 46 has aberrant effects on mitotic spindle orientation, and that HTT is required for normal distribution of mitotic components, arguing for allele-specific silencing as a therapy. This work also demonstrates that using physiologically relevant CAG-repeat lengths (between 36-60) can be important when modeling HD.
Another way to optimize allele-specific silencing is by optimizing the chemistry of oligonucleotides. Thiophosphonacetate (thio-PACE) modifications replace a non-bridging oxygen molecule with an acetate and a phosphorothiate substitution, modifications hypothesized to increase uptake and hybridization of the oligonucleotide with its target. A recent article describes the delivery of these altered oligonucleotides to fibroblasts derived from HD patients and to an immortalized striatal cell model.3 Although oligonucleotides with different numbers of these modifications were able to silence mHTT, not all oligonucleotides had positive results compared to the unmodified control. These results allude to the importance of, and potential to modify, the chemistry of oligonucleotides for silencing mHTT.
1Fink KD, Deng P, Gutierrez J, et al. Allele-specific reduction of the mutant huntingtin allele using transcription activator-like effectors in human Huntington’s disease fibroblasts. Cell Transplant. 2016;25(4):677-686.
2Lopes C, Aubert S, Bourgois-Rocha F, et al. Dominant-negative effects of adult-onset huntingtin mutations alter the division of human embryonic stem cells-derived neural cells. PloS one. 2016;11(2):e0148680.
3Matsui M, Threlfall RN, Caruthers MH, Corey DR. Effect of 2′-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells. Artif DNA PNA XNA. 2014;5(3):e1146391.