Journal of Huntington's Disease

Cerebrospinal fluid (CSF) is enriched in brain-derived components and represents an accessible and appealing means of interrogating the CNS milieu to study neurodegenerative diseases and identify biomarkers to facilitate the development of novel therapeutics. Many such CSF biomarkers have been proposed for Huntington’s disease (HD) but none has been validated for clinical trial use. Across many studies proposing dozens of biomarker candidates, there is a notable lack of statistical power, consistency, rigor and validation. Here we review proposed CSF biomarkers including neurotransmitters, transglutaminase activity, kynurenine pathway metabolites, oxidative stress markers, inflammatory markers, neuroendocrine markers, protein markers of neuronal death, proteomic approaches and mutant huntingtin protein itself. We reflect on the need for large-scale, standardized CSF collections with detailed phenotypic data to validate and qualify much-needed CSF biomarkers for clinical trial use in HD.

Dysphagia is a very common occurrence in Huntington’s disease (HD). As such, many people with HD require texture modified diets. This commentary discusses the implications for individuals living long-term on modified diets, including the loss of sensory stimulation and dietary enjoyment. Clinical practice analyses of two interventions aimed at promoting dietary satisfaction and involvement in food preparation for those with HD are described and parameters for future research are discussed.

Background: Impairments in energy metabolism are implicated in Huntington’s disease (HD) pathogenesis. Reduced levels of the mitochondrial enzyme succinate dehydrogenase (SDH), the main element of complex II, are observed post mortem in the brains of HD patients, and energy metabolism defects have been identified in both presymptomatic and symptomatic HD patients.

Objective: Chemical preconditioning with 3-nitropropionic acid (3-NP), an irreversible inhibitor of SDH, has been shown to increase tolerance against experimental hypoxia in both heart and brain. Here we studied the effect of chronic preconditioning in the R6/2 mouse model of HD using mice carrying CAG repeat lengths of either 250 or 400 repeats. Both are transgenic fragment models, with 250CAG mice having a more rapid disease progression than 400CAG mice.

Methods: Low doses of 3-NP (24 mg/kg) were administered via the drinking water and the effect on phenotype progression and cognition function assessed.

Results: After 3-NP treatment there were significant improvements in all aspects of the behavioural phenotype, apart from body weight, with timing and magnitude of improvements dependent on both CAG repeat length and sex. Specifically, a delay in the deterioration of general health (as shown by delayed onset of glycosuria and increased survival) was seen in both male and female 400CAG mice and in female 250CAG mice and was consistent with improved appearance of 3-NP treated R6/2 mice. Male 250CAG mice showed improvements but these were short term, and 3-NP treatment eventually had deleterious effects on their survival rate. When cognitive performance of 250CAG mice was assessed using a two-choice discrimination touchscreen task, we found that female mice showed significant improvements.

Discussion: Together, our results support the idea that energy metabolism contributes to the pathogenesis of HD, and suggest that improving energy deficits might be a therapeutically useful target.

Background: Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington’s disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments.

Objectives: We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA.

Methods: Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection.

Results: Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites.

Conclusions: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.

Background: The Huntington’s disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation.

Objective: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt.

Methods: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq.

Results: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3’ UTR.

Conclusions: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases.

Background: Dysregulation of iron homeostasis is implicated in the pathogenesis of Huntington’s disease. We have previously shown that increased iron intake in R6/2 HD neonatal mice, but not adult R6/2 HD mice potentiates disease outcomes at 12-weeks of age corresponding to advanced HD [Redox Biol. 2015;4 : 363–74]. However, whether these findings extend to other HD models is unknown. In particular, it is unclear if increased neonatal iron intake can promote neurodegeneration in mouse HD models where disease onset is delayed to mid-adult life.

Objective: To determine if increased dietary iron intake in neonatal and adult life-stages potentiates HD in the slowly progressive YAC128 HD mouse model.

Methods: Female neonatal mice were supplemented daily from days 10–17 with 120μg/g body weight of carbonyl iron. Adult mice were provided diets containing low (50 ppm), medium (150 ppm) and high (500 ppm) iron concentrations from 2-months of age. HD progression was determined using behavioral, brain morphometric and biochemical approaches.

Results: Neonatal-iron supplemented YAC128 HD mice had significantly lower striatal volumes and striatal neuronal cell body volumes as compared to control HD mice at 1-year of age. Neonatal-iron supplementation of HD mice had no effect on rota-rod motor endurance and brain iron or glutathione status. Adult iron intake level had no effect on HD progression. YAC128 HD mice had altered peripheral responses to iron intake compared to iron-matched wild-type controls.

Conclusions: Female YAC128 HD mice supplemented with nutritionally-relevant levels of iron as neonates demonstrate increased striatal degeneration 1-year later.

Background: Huntington’s disease (HD) is a fatal, inherited neurodegenerative disorder characterized by uncontrollable dance-like movements, as well as cognitive deficits and mood changes. A feature of HD is a metabolic disturbance that precedes neurological symptoms. In addition, brain cholesterol synthesis is significantly reduced, which could hamper synaptic transmission.

Objective: Alterations in lipid metabolism as a potential target for therapeutic intervention in the R6/2 mouse model of HD were examined.

Methods: Electrophysiological recordings in vitro examined the acute effects of cholesterol-modifying drugs. In addition, behavioral testing, effects on synaptic activity, and measurements of circulating and brain tissue concentrations of cholesterol and the ketone β-hydroxybutyrate (BHB), were examined in symptomatic R6/2 mice and littermate controls raised on normal chow or a ketogenic diet (KD).

Results: Whole-cell voltage clamp recordings of striatal medium-sized spiny neurons (MSNs) from symptomatic R6/2 mice showed increased frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) compared with littermate controls. Incubation of slices in cholesterol reduced the frequency of large-amplitude sIPSCs. Addition of BHB or the Liver X Receptor (LXR) agonist T0901317 reduced the frequency and amplitude of sIPSCs. Surprisingly, incubation in simvastatin to reduce cholesterol levels also decreased the frequency of sIPSCs. HD mice fed the KD lost weight more gradually, performed better in an open field, had fewer stereotypies and lower brain levels of cholesterol than mice fed a regular diet.

Conclusions: Lipid metabolism represents a potential target for therapeutic intervention in HD. Modifying cholesterol or ketone levels acutely in the brain can partially rescue synaptic alterations, and the KD can prevent weight loss and improve some behavioral abnormalities.

Background: REGISTRY is the largest European observational study of Huntington’s disease (HD). The Leiden University Medical Center (LUMC) in The Netherlands is the largest recruiting site.

Objective: The aim of this paper is to give an overview of the baseline characteristics of all Leiden participants from the start of the study in 2005 until the close of REGISTRY at the LUMC in September 2014.

Methods: The Leiden cohort is described in two different ways: CAG repeat length and presence of motor signs.

Results: Division into groups based on prolonged CAG length revealed that the cohort consists of 4 intermediate - (27–35 CAG), 22 reduced penetrance - (36–39 CAG), 465 full penetrance - (>39 CAG) and 60 control participants (<27 CAG). The second way of dividing the participants based on present or absent of motor signs, showed that 170 pre-motormanifest - and 317 motormanifest participants were enrolled.

Conclusion: The Leiden REGISTRY cohort at baseline is mainly characterized by full penetrance gene expansion carriers who have been clinically diagnosed with HD but who remain relatively functionally independent. For the majority of these participants, disease onset was based on motor signs followed by psychiatric and cognitive signs.

Background: Visuospatial deficits have been described in Huntington’s disease (HD); however, the extent of these deficits remains unclear. The Benton Judgment of Line Orientation (JoLO) Test, commonly used to assess visuospatial ability, requires minimal motor involvement. It has demonstrated sensitivity to visuospatial deficits in Parkinson’s disease; however, few studies have examined performance on this test in HD.

Objective: The objective of the current study was to assess visuospatial ability in premanifest and manifest HD using the JoLO.

Methods: A global cognitive measure, the Mattis Dementia Rating Scale (DRS), was used to stratify manifest HD patients as mild (DRS ≥129) vs. moderate-severe (DRS ≤128). Fifty mild, 42 moderate-severe, and 30 premanifest HD subjects, as well as 35 matched controls, were administered the JoLO. HD Burden of Pathology (BOP) scores were used as a measure of disease severity.

Results: Results revealed that the total manifest HD sample (p <  0.001), in addition to the mild (p = 0.028), and moderate-severe (p <  0.001), but not premanifest, HD subjects scored significantly lower on the JoLO compared to normal controls.

Conclusions: Our results suggest that the JoLO is useful for detecting visuospatial deficits across various stages of manifest HD. However, any visuospatial impairment that might be present during the premanifest stage of HD was not detected using the JoLO in the present sample.