Andreas Weiss

Sustained Therapeutic Reversal of Huntington's Disease by Transient Repression of Huntingtin Synthesis

The primary cause of Huntingtons disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Proteolysis of Mutant Huntingtin Produces an Exon 1 Fragment That Accumulates as an Aggregated Protein in Neuronal Nuclei in Huntington Disease

Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.

Altered chromatin architecture underlies progressive impairment of the heat shock response in mouse models of Huntington disease

Huntington disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. Previous studies have proposed that activation of the heat shock response (HSR) via the transcription factor heat shock factor 1 (HSF1) may be of therapeutic benefit. However, the effect of disease progression on the HSR and the therapeutic potential of this pathway are currently unknown. Here, we used a brain-penetrating HSP90 inhibitor and physiological, molecular, and behavioral readouts to demonstrate that pharmacological activation of HSF1 improves huntingtin aggregate load, motor performance, and other HD-related phenotypes in the R6/2 mouse model of HD. However, the beneficial effects of this treatment were transient and diminished with disease progression. Molecular analyses to understand the transient nature of these effects revealed altered chromatin architecture, reduced HSF1 binding, and impaired HSR accompanied disease progression in both the R6/2 transgenic and HdhQ150 knockin mouse models of HD. Taken together, our findings reveal that the HSR, a major inducible regulator of protein homeostasis and longevity, is disrupted in HD. Consequently, pharmacological induction of HSF1 as a therapeutic approach to HD is more complex than was previously anticipated.

Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington's disease

A CAG‐repeat gene expansion translated into a pathogenic polyglutamine stretch at the N‐terminus of huntingtin triggers Huntington’s Disease. Mutated huntingtin is predicted to adopt toxic properties mainly if aggregation‐prone N‐terminal fragments are released by proteolysis. Huntingtin‐aggregates are indeed a major hallmark of this disorder and could represent useful markers of disease‐onset or progression. We designed a simple method for qualitative and quantitative characterization of aggregates. For this, we analyzed samples from in vitro and in vivo Huntington’s Disease models by agarose gel electrophoresis and showed that in the brain of transgenic mice huntingtin‐aggregates became larger as a function of disease progression. This appears to be a property of cytoplasmic but not nuclear aggregates. In cell cultures, treatment with Congo Red inhibited aggregate growth but not total load. Finally, we showed that in primary striatal neurons and in brains of R6/2 and HdhQ150 mice, the presence of aggregates preceded initiation of any other functional deficits. This observation argues for a pathogenic role of huntingtin‐aggregation in Huntington’s Disease. Our results emphasize that thorough analysis of huntingtin metabolism and aggregation is now feasible, thus significantly improving the power of studies assessing therapies designed to lower huntingtin levels or to interfere with its aggregation.

Single-step detection of mutant huntingtin in animal and human tissues: a bioassay for Huntington's disease.

The genetic mutation causing Huntingtons disease is a polyglutamine expansion in the huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant huntingtin, understanding huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Förster resonance energy transfer (FRET) assay for quantification of soluble mutant huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant huntingtin. The design of the homogeneous one-step method for huntingtin detection is such that it can be easily applied to measure other proteins of interest.

SIRT2 Ablation Has No Effect on Tubulin Acetylation in Brain, Cholesterol Biosynthesis or the Progression of Huntington's Disease Phenotypes In Vivo

Huntingtons disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. The molecular pathogenesis of HD is complex and many mechanisms and cellular processes have been proposed as potential sites of therapeutic intervention. However, prior to embarking on drug development initiatives, it is essential that therapeutic targets can be validated in mammalian models of HD. Previous studies in invertebrate and cell culture HD models have suggested that inhibition of SIRT2 could have beneficial consequences on disease progression. SIRT2 is a NAD+-dependent deacetylase that has been proposed to deacetylate α-tubulin, histone H4 K16 and to regulate cholesterol biogenesis – a pathway which is dysregulated in HD patients and HD mouse models. We have utilized mice in which SIRT2 has been reduced or ablated to further explore the function of SIRT2 and to assess whether SIRT2 loss has a beneficial impact on disease progression in the R6/2 mouse model of HD. Surprisingly we found that reduction or loss of SIRT2 had no effect on the acetylation of α-tubulin or H4K16 or on cholesterol biosynthesis in the brains of wild type mice. Equally, genetic reduction or ablation of SIRT2 had no effect on HD progression as assessed by a battery of physiological and behavioural tests. Furthermore, we observed no change in aggregate load or levels of soluble mutant huntingtin transprotein. Intriguingly, neither the constitutive genetic loss nor acute pharmacological inhibition of SIRT2 affected the expression of cholesterol biosynthesis enzymes in the context of HD. Therefore, we conclude that SIRT2 inhibition does not modify disease progression in the R6/2 mouse model of HD and SIRT2 inhibition should not be prioritised as a therapeutic option for HD.

HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration

HDAC4 histone deacetylase is found to associate with huntingtin in a polyQ-length dependent manner. Reduction of HDAC4 levels in mouse models of Huntingtons disease (HD) delays cytoplasmic aggregation in the brain and improves the molecular pathology of HD, providing a potential new therapeutic target.

HTT-lowering reverses Huntington’s disease immune dysfunction caused by NFκB pathway dysregulation

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntingtons disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntingtons disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntingtons disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntingtons disease.

Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression

Huntingtons disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.

Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients

BACKGROUND Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntingtons disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. METHODS We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. RESULTS This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. CONCLUSIONS We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD. TRIAL REGISTRATION Not applicable. FUNDING CHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.