Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway

Huntington’s disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein–RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.     

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150^Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150^Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150^Glued complex interactions.     

N-alpha-acetylation of Huntingtin protein increases its propensity to aggregate

Huntington’s disease (HD) is a neurodegenerative disorder caused by a poly-CAG expansion in the first exon of the HTT gene, resulting in an extended poly-glutamine tract in the N-terminal domain of the Huntingtin (Htt) protein product. Proteolytic fragments of the poly-glutamine–containing N-terminal domain form intranuclear aggregates that are correlated with HD. Post-translational modification of Htt has been shown to alter its function and aggregation properties. However, the effect of N-terminal Htt acetylation has not yet been considered. Here, we developed a bacterial system to produce unmodified or N-terminally acetylated and aggregation-inducible Htt protein. We used this system together with biochemical, biophysical, and imaging studies to confirm that the Htt N-terminus is an in vitro substrate for the NatA N-terminal acetyltransferase and show that N-terminal acetylation promotes aggregation. These studies represent the first link between N-terminal acetylation and the promotion of a neurodegenerative disease and implicates NatA-mediated Htt acetylation as a new potential therapeutic target in HD.     

Longitudinal Study of Informed Consent in Innovative Therapy Research: Experience and Provisional Recommendations from a Multicenter Trial of Intracerebral Grafting

BackgroundThere is an urgent need to assess and improve the consent process in clinical trials of innovative therapies for neurodegenerative disorders.MethodsWe performed a longitudinal study of the consent of Huntington’s disease patients during the Multicenter Fetal Cell Intracerebral Grafting Trial in Huntington’s Disease (MIG-HD) in France and Belgium. Patients and their proxies completed a consent questionnaire at inclusion, before signing the consent form and after one year of follow-up, before randomization and transplantation. The questionnaire explored understanding of the protocol, satisfaction with the information delivered, reasons for participating in the trial and expectations regarding the transplant. Forty-six Huntington’s disease patients and 27 proxies completed the questionnaire at inclusion, and 27 Huntington’s disease patients and 16 proxies one year later.ResultsThe comprehension score was high and similar for Huntington’s disease patients and proxies at inclusion (72.6% vs 77.8%; P > 0.1) but only decreased in HD patients after one year. The information satisfaction score was high (73.5% vs 66.5%; P > 0.1) and correlated with understanding in both patients and proxies. The motivation and expectation profiles were similar in patients and proxies and remained unchanged after one year.ConclusionsCognitively impaired patients with Huntington’s disease were capable of consenting to participation in this trial. This consent procedure has presumably strengthened their understanding and should be proposed before signing the consent form in future gene or cell therapy trials for neurodegenerative disorders. Because of the potential cognitive decline, proxies should be designated as provisional surrogate decision-makers, even in competent patients.     

High Protein Diet and Huntington's Disease

Huntington’s disease (HD) is a neurodegenerative disorder caused by the huntingtin (HTT) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (p = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.     

A Broad Phenotypic Screen Identifies Novel Phenotypes Driven by a Single Mutant Allele in Huntington’s Disease CAG Knock-In Mice

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.     

Huntingtin Is Critical Both Pre- and Postsynaptically for Long-Term Learning-Related Synaptic Plasticity in Aplysia

Patients with Huntington’s disease exhibit memory and cognitive deficits many years before manifesting motor disturbances. Similarly, several studies have shown that deficits in long-term synaptic plasticity, a cellular basis of memory formation and storage, occur well before motor disturbances in the hippocampus of the transgenic mouse models of Huntington’s disease. The autosomal dominant inheritance pattern of Huntington’s disease suggests the importance of the mutant protein, huntingtin, in pathogenesis of Huntington’s disease, but wild type huntingtin also has been shown to be important for neuronal functions such as axonal transport. Yet, the role of wild type huntingtin in long-term synaptic plasticity has not been investigated in detail. We identified a huntingtin homolog in the marine snail Aplysia, and find that similar to the expression pattern in mammalian brain, huntingtin is widely expressed in neurons and glial cells. Importantly the expression of mRNAs of huntingtin is upregulated by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia. Furthermore, we find that huntingtin expression levels are critical, not only in presynaptic sensory neurons, but also in the postsynaptic motor neurons for serotonin-induced long-term facilitation at the sensory-to-motor neuron synapse of the Aplysia gill-withdrawal reflex. These results suggest a key role for huntingtin in long-term memory storage.     

The Sirtuin-2 Inhibitor AK7 Is Neuroprotective in Models of Parkinson’s Disease but Not Amyotrophic Lateral Sclerosis and Cerebral Ischemia

Sirtuin deacetylases regulate diverse cellular pathways and influence disease processes. Our previous studies identified the brain-enriched sirtuin-2 (SIRT2) deacetylase as a potential drug target to counteract neurodegeneration. In the present study, we characterize SIRT2 inhibition activity of the brain-permeable compound AK7 and examine the efficacy of this small molecule in models of Parkinson’s disease, amyotrophic lateral sclerosis and cerebral ischemia. Our results demonstrate that AK7 is neuroprotective in models of Parkinson’s disease; it ameliorates alpha-synuclein toxicity in vitro and prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion and dopaminergic neuron loss in vivo. The compound does not show beneficial effects in mouse models of amyotrophic lateral sclerosis and cerebral ischemia. These findings underscore the specificity of protective effects observed here in models of Parkinson’s disease, and previously in Huntington’s disease, and support the development of SIRT2 inhibitors as potential therapeutics for the two neurodegenerative diseases.     

A Huntingtin Peptide Inhibits PolyQ-Huntingtin Associated Defects

Background

Huntington’s disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects.

Methodology/Principal Findings

The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington’s disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias.

Conclusions/Significance

Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington’s disease.     

How to Capitalize on the Retest Effect in Future Trials on Huntington’s Disease

The retest effect—improvement of performance on second exposure to a task—may impede the detection of cognitive decline in clinical trials for neurodegenerative diseases. We assessed the impact of the retest effect in Huntington’s disease trials, and investigated its possible neutralization. We enrolled 54 patients in the Multicentric Intracerebral Grafting in Huntington’s Disease (MIG-HD) trial and 39 in the placebo arm of the Riluzole trial in Huntington’s Disease (RIL-HD). All were assessed with the Unified Huntington’s Disease Rating Scale (UHDRS) plus additional cognitive tasks at baseline (A₁), shortly after baseline (A₂) and one year later (A₃). We used paired t-tests to analyze the retest effect between A₁ and A₂. For each task of the MIG-HD study, we used a stepwise algorithm to design models predictive of patient performance at A₃, which we applied to the RIL-HD trial for external validation. We observed a retest effect in most cognitive tasks. A decline in performance at one year was detected in 3 of the 15 cognitive tasks with A₁ as the baseline, and 9 of the 15 cognitive tasks with A₂ as the baseline. We also included the retest effect in performance modeling and showed that it facilitated performance prediction one year later for 14 of the 15 cognitive tasks. The retest effect may mask cognitive decline in patients with neurodegenerative diseases. The dual baseline can improve clinical trial design, and better prediction should homogenize patient groups, resulting in smaller numbers of participants being required.