The pool of free intracellular Mg2+ varies among tissues with the highest concentration measured in muscle tissue and the lowest measured in immune cells and in the brain. Here we investigate the impact of free Mg2+ on the fidelity of human DNA ligase I (LIG1). LIG1 is the major DNA ligase and is required to complete DNA replication, recombination and repair pathways. Biallelic hypomorphic variants of LIG1 cause immunodeficiency-96. We employed steady-state kinetics to compare fidelity of LIG1 towards a damaged nucleobase at the 3'- hydroxyl side of a nicked DNA substrate. The fidelity for discrimination between a damaged and undamaged nick increases by 21-fold when the free Mg2+ concentration is decreased from 1.0 to 0.2 mM. This has important implications for neurodegenerative and immune diseases, because the brain and the immune system are reported to have free Mg2+ concentration in the range from 0.2 to 0.4 mM. We examined a recently characterized minor variant of LIG1, K845N, which has a protective effect in Huntington's disease, and found that the fidelity of K845N LIG1 is also enhanced as free Mg2+ decreases. This increase in fidelity is mainly due to the increased release of the AMP-DNA intermediate from a pro-mutagenic DNA substrate. A model is proposed whereby the fidelity of DNA transactions is sensitive to the availability of the Mg2+ cofactor for DNA ligation and therefore ligation fidelity may vary between tissues.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by inheriting an expanded CAG repeat tract in the huntingtin gene (HTT) that further expands in somatic cells over an individual's lifetime. Genome-wide association studies have provided critical insight into factors that modify the course of disease. These include DNA repair genes that alter the rate of somatic expansion and other genes that do not appear to directly influence this process. One modifier gene is DNA ligase 1 (LIG1), in which a variant specifying a lysine to asparagine substitution (K845N) is associated with a profound (7 to 8 y) delay in the onset of motor signs. Here, we have taken a multifaceted approach to gain insight into the protective nature of this variant in HD. We demonstrate using in vitro ligase assays and enzyme kinetics that K845N enhances discrimination toward mismatched substrates and increases repair fidelity. Consistent with increased ligation fidelity, K845N confers protection against oxidative stress in cell-based assays. Finally, we demonstrate that the mouse LIG1 K843N orthologue suppresses somatic CAG expansion in HD knock-in mice. Overall, our data provide evidence that altered LIG1 function due to the K845N substitution may contribute to HD clinical delay by slowing somatic expansion in the brain and protecting the genome globally against damage. Significantly, our results provide a mechanistic foundation for considering DNA ligase fidelity as a therapeutic target in HD and potentially in other trinucleotide repeat disorders.

Huntington's disease (HD) is driven by somatic expansion of the HTT CAG repeat, with onset modified by genetic factors. One such modifier, 8AM1, maps to chromosome 8 near RRM2B, a gene not directly involved in the machinery that lengthens the repeat. To investigate this locus, we performed capture sequencing and identified variants at both the 5' and 3' ends of RRM2B with expected minor allele frequencies. A polymorphic frameshift variant (rs1037699) in an alternate exon 1 disrupts expression of a previously uncharacterized RRM2B isoform 2, but not isoform 1. Functional analyses in RRM2B knock-out cells and 8AM1 heterozygous LCLs suggest that isoform 2 may function at mitochondria. Several 3' variants, including a 21 bp 3'UTR deletion (rs200678743) and peak tag-SNV (rs79136984), act as cis expression quantitative trait loci. Analysis of HD onset data (n = 12,982) revealed that 5' and 3' variants contribute independently to the 8AM1 modifier effect, with full impact observed only in the absence of the frameshift variant. Knockdown of both isoforms increased neurodegeneration in HD neurons derived from pre-symptomatic patient fibroblasts, supporting an intersection of RRM2B biology and HD pathogenesis. We conclude that the 8AM1 haplotype, present in ~ 14% of Europeans, modifies RRM2B expression in a cell- and context-dependent manner, thereby accelerating HD onset in mutation carriers.

Huntington’s disease (HD) is a neurodegenerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phosphosites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein by proteomic and phosphoproteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phosphosites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together, these findings highlight categories of phosphosites that merit further study and provide a phosphosite kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

Dominant gain-of-function mechanisms in Huntington’s disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif–altering (PAM-altering) SNP (PAS), we developed an allele-specific CRISPR/Cas9 strategy to permanently inactivate mutant HTT through nonsense-mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant-specific CRISPR/Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR/Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR/Cas9 strategy inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR/Cas9 and supporting its therapeutic potential in HD.

Genome-wide association studies (GWASs) of Huntington disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction by using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWASs. Combined with imputation with the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified additional modifier effects at the PMS1 and PMS2 loci and implicated a potential second locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.

Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the polyglutamine (polyQ) expansion in huntingtin (HTT) protein. The challenge of obtaining full-length HTT proteins with high purity limits the understanding of the HTT protein function. Here, we provide a protocol to generate and purify full-length recombinant human HTT proteins with various polyQ lengths, which is key to investigate the biochemical function of HTT proteins and the molecular mechanism underlying HD pathology. For complete details on the use and execution of this protocol, please refer to Jung et al. (2020).

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT:Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT. These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3′-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3′-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3′-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3′-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3′-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

Rare individuals with inactivating mutations in the Huntington's disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development.

AbstractThe neurodegenerative Huntington’s disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pmand optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cellsin vitroand in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.

We present a new measurement of the CKM matrix element $|V_{cb}|$ from $ B^{0} rightarrow D^{*}ell nu$ decays, reconstructed with full Belle data set ($711 , rm fb^{-1}$). Two form factor parameterisations, based on work by the CLN and BGL groups, are used to extract the product $mathcal{F}(1)eta_{rm EW}|V_{cb}|$ and the decay form factors, where $mathcal{F}(1)$ is the factor normalisation and $eta_{rm EW}$ is a small electroweak correction. In the CLN parameterisation we find $mathcal{F}(1)eta_{rm EW}|V_{cb}| = (35.06 pm 0.15 pm 0.54) times 10^{-3}$, $rho^{2}=1.106 pm 0.031 pm 0.007$, $R_{1}(1)=1.229 pm 0.028 pm 0.009$, $R_{2}(1)=0.852 pm 0.021 pm 0.006$. In the BGL parameterisation we find $mathcal{F}(1)eta_{rm EW}|V_{cb}|= 38.73 pm 0.25 pm 0.60$, which is higher but consistent with the determination from inclusive semileptonic $B$ decays when correcting for $mathcal{F}(1)eta_{rm EW}$. This is the most precise measurement of $mathcal{F}(1)eta_{rm EW}|V_{cb}|$ and form factors that has ever been carried out, and the first direct study of the BGL form factor parameterisation in an experimental measurement.

The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington’s disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the C-terminal CTD-II domain. Aptamer binding to mutant huntingtin abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPCs), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPCs against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cishaplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.