Components of the DNA mismatch repair (MMR) pathway modulate somatic CAG-repeat expansion at the huntingtin (HTT) locus and alter the age of clinical motor diagnosis and progression of Huntington's disease (HD). MSH3, which encodes a subunit of MutSβ (MSH2-MSH3), has been identified as a genetic modifier of age at onset of multiple clinical phenotypes in people with HD (PwHD). MutSβ interacts with MutL endonucleases (MutLα, MLH1-PMS2 and MutLγ MLH1-MLH3) in a ternary complex with DNA to initiate MMR. Disruption of the MLH1-MSH3 protein-protein interaction (PPI) represents a potential therapeutic strategy to inhibit MMR and limit somatic CAG-repeat expansion. Leveraging the MLH1 Interaction Protein (MIP) Box sequence and the structural information of the hMLH1 C-terminal domain (CTD) bound to an MSH3-derived peptide (21 QAVLSRFFQ 29), we designed and optimized potent linear and cyclic peptides targeting the MLH1-MSH3 interface. Dimerization of the optimized cyclic peptide (compound 52) enhanced binding affinity, achieving double-digit nanomolar potency in a MutSβ:MutLα:DNA (MLH1-MSH3-DNA) ternary complex assay in the presence of 100 μM of ATP (compound 52 IC50 = 87 nM, compound 56 IC50 = 32 nM). Evaluation in an in vitro MMR DNA repair assay using cellular nuclear extracts from MMR-proficient and-deficient cell lines demonstrated that our lead compound (56) effectively inhibits MutSβ/MutLα-dependent DNA repair in human cells. These results highlight cyclic peptide-mediated disruption of MLH1-MSH3 as a promising approach to modulate MMR activity and possibly mitigate somatic CAG-repeat expansion in HD.

Aggregation of mutant huntingtin (mHTT) is a neurologic hallmark of Huntington disease (HD), a neurodegenerative disorder caused by the expansion of a cytosine-adenine-guanine repeat tract in the huntingtin gene (HTT). With a considerable number of candidate therapeutic interventions aimed at lowering mHTT expression under investigation, noninvasive monitoring of changes in mHTT aggregate levels in the brain could hasten the development and identification of disease-modifying therapies. Here we evaluate a new radioligand, [18F]CHDI-385, to quantify mHTT aggregates using microPET imaging in the zQ175DN mouse model of HD. Methods: In 3- and 9-mo old wild-type (n = 24 for each age) and heterozygous zQ175DN (n = 24 for each age) mice, we assessed the plasma and brain radiometabolite profile, explored in vivo tracer kinetics (including test-retest variability), and performed quantitative (using total volume of distribution based on a noninvasive image-derived input function, 0-120 min) and semiquantitative (using SUV; time interval, 100-120 min after injection) analyses to determine the performance of this radioligand in detecting mHTT aggregates in vivo. Results: [18F]CHDI-385 showed metabolic stability in both wild-type and heterozygous mice as well as sufficient cerebral retention time in both genotypes. Quantitative (2-tissue compartmental model and Logan graphical analysis) and semiquantitative (SUV) analyses were in strong agreement with one another (striatum, r2 = 0.986; P < 0.0001). Differences in measures of [18F]CHDI-385 uptake were significant between heterozygous mice and wild-type mice at both 3 mo (P < 0.001) and 9 mo (P < 0.0001). In addition, [18F]CHDI-385 displayed a good to excellent test-retest variability as indicated by the intraclass correlation coefficient (ICC) with both quantitative (ICC, 0.62-0.78) and semiquantitative (ICC, 0.65-0.80) analyses. Conclusion: [18F]CHDI-385 demonstrated excellent kinetics and reliable semiquantitative and quantitative performance. Importantly, the validation of semiquantitative analysis supports the use of the more clinically friendly SUV metric, which does not require the use of an input function and metabolite correction. These results indicate that [18F]CHDI-385 is a radioligand with optimal properties for detecting and quantifying cerebral mHTT aggregates and support its clinical evaluation.

Huntington's disease is caused by a CAG repeat tract expansion in the huntingtin gene, resulting in production of pathogenic N-terminal huntingtin protein fragments associated with disease pathology. Despite their central role, detection of these fragments has relied on a limited antibody repertoire with reproducibility concerns. Here, we describe the generation and characterization of recombinant rabbit monoclonal antibodies targeting two reciprocal neoepitopes flanking the huntingtin exon 1/exon 2 junction corresponding to amino acids P90 and K91. The P90 antibodies (clones 1B12, 11G2) demonstrate fragment-length-selective recognition of the C-terminal HTTexon1 P90 neoepitope with no detectable binding to full length huntingtin. A side-by-side comparison of the widely used monoclonal antibody MW8 from two different sources revealed measurable lot-to-lot drift in its fragment selectivity, whereas the recombinant P90 antibodies, expressed from a defined, sequenced clone, maintained consistent specificity, addressing this source-dependent variability. Whereas P90-positive fragments can arise through alternative splicing of the HTT1a transcript, generation of the reciprocal K91 N-terminal HTTexon2 neoepitope would require site-specific proteolytic cleavage, a mechanism that has not yet been directly tested for lack of a suitable reagent. The K91 antibody (clone 7G10) binds the N-terminal K91 neoepitope with high affinity and specificity over full length huntingtin and provides, for the first time, a tool capable of directly interrogating whether such cleavage occurs. Neoepitope specificity of these antibodies was orthogonally confirmed by protease digestion (Lys-N and Arg-C) coupled with intact mass spectrometry. As an additional outcome of the immunization and selection strategy, we discovered human-mouse cross-reactive antibodies (clones 27F5, 31C10) targeting the proline-rich domain of huntingtin that will facilitate mouse-human translational studies. All antibodies are recombinant, ensuring long-term reproducibility, and are being made available, along with their sequences, to the research community.

Orthosteric inhibitors of the human heterodimeric DNA mismatch repair complex MutSβ were identified by high-throughput screening. Following extensive hit confirmation to remove false positives, two series were found to give consistent activity free of likely artefactual effects. Extensive hit profiling confirmed an ATP-competitive mode of action and resulted in our obtaining the first reported X-ray and cryo-EM structures of small molecule inhibitors of MutSβ occupying the ATP-binding site of MSH3.

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide repeat in the huntingtin gene (HTT) that subsequently leads to aggregation of the mutant huntingtin (mHTT) protein. Thus, lowering mHTT is a key therapeutic approach used by several candidate therapeutics currently under investigation. Visualization of the efficiency of these therapeutics through in vivo mHTT quantification rises in importance. For positron emission tomography (PET) imaging of mHTT aggregates, it is critical to characterize the in vivo kinetic profile of newly identified mHTT binders to assess their translational application. Here, we report the evaluation of [11C]CHDI-009R, a PET imaging radioligand with higher affinity and selectivity for mHTT aggregates than previously reported radioligands, in the heterozygous zQ175DN mouse model of HD and wild-type littermates at 9 and 3 months of age. [11C]CHDI-009R displayed high stability in plasma and brain, which was reflected in brain kinetics as demonstrated by rapid uptake followed by relatively slow elimination. Kinetic modeling and volume of distribution VT (IDIF) indicated the radioligand's ability to quantify mHTT aggregation at 9 months of age with clear genotype differentiation (p < 0.0001). [11C]CHDI-009R showed an excellent test–retest reliability in 9-month-old mice (intraclass correlation coefficient: 0.62—0.79). A phenotypic difference in mHTT aggregates was also observed in 3-month-old mice in several brain structures (p < 0.05) and was confirmed with [3H]CHDI-009R autoradiography. Overall, this study suggests [11C]CHDI-009R is a promising radioligand for the detection of cerebral mHTT aggregates in a mouse model of HD and supports its advance to clinical evaluation.

Huntington's disease (HD) is caused by the repeat expansion of the CAG trinucleotide in the mutant Huntingtin gene (mHTT) within the exon1 region, resulting in an expanded polyglutamine-containing mHTT exon1 protein that serves as the source of the hallmark mHTT aggregates in people with HD (PwHD). To better understand aggregation formation during disease progression and its utility as a pharmacodynamic biomarker, we have been targeting mHTT aggregates for developing PET imaging tracers and have identified a series of isoindolinones that show significantly higher binding potential (BP, a ratio of Bmax over KD) in HD mouse models as well as increased binding in HD post-mortem brains, compared to first generation ligands. We present the structure-activity relationship (SAR) work leading to three candidate tracers progressed for human studies: [11C]CHDI-009 (6), [18F]CHDI-385 (29) and [18F]CHDI-386 (30).

Huntington's disease is a neurodegenerative disorder associated with a polyglutamine expansion within the first exon of the huntingtin protein (HTT exon 1). This mutation results in HTT dysfunction and the production of N-terminal HTT aggregates. The dimerization of the HTT exon 1 fragment through self-association of the first 17 residues (N17) is considered the initial step in the HTT exon 1 aggregation pathway. The association of N17 with membranes has been proposed to catalyze aggregation by increasing the local concentration of exon 1, and post-translational modifications (PTMs) in N17 are known to influence membrane interaction and the aggregation rate of exon 1. To elucidate the influence of N17 PTMs on both self-association and membrane interaction, thereby gaining insight into HTT function and exon 1 aggregation, we used solution nuclear magnetic resonance and circular dichroism spectroscopies to address loss of initial methionine, subsequent acetylation, and phosphorylation of threonine and serines. Our findings indicate that modifications to N17 that enhance helicity correspond to increased self-association and membrane interaction. We then conducted X-ray crystallographic studies that led to a proposed HTT exon 1 dimerization model consistent with the association of N17 dimers. This provides insight into the impact of PTMs on HTT aggregation. The experimental methods and N17 self-association model we describe may serve as a foundation for further experiments exploring the influence of N17 PTMs on HTT function and pathogenicity.

Huntington’s disease (HD) is a progressive neurodegenerative disease caused by the pathologic expansion of a CAG repeat in the first exon of the huntingtin ( HTT ) gene, resulting in a huntingtin (HTT) protein with an expanded polyglutamine (polyQ) tract. Phosphorylation at residue S421 (pS421) is one of the post-translational modifications proposed to influence the biology of wild-type and mutant (m)HTT, such as HTT stability and clearance, HTT subcellular localization, mHTT toxicity, and regulation of HTT function in axonal transport. However, the detection and quantification of S421-HTT phosphorylation in relevant biological contexts have remained challenging and the consequences of pS421 in HD pathogenesis remains unclear. Here we report the development of a novel ultrasensitive immunoassay enabling the specific and sensitive detection of pS421-HTT in a variety of biologically relevant contexts. With this assay we conducted a longitudinal assessment of pS421 levels in tissues from a mouse model of HD to investigate the relationship between S421 phosphorylation and phenotypic progression. We also identified PRKACA, the cAMP-regulated catalytic α subunit of PKA, as a kinase capable of phosphorylating S421-HTT, demonstrating its ability to regulate endogenous pS421 in human cells. Finally, we exploited the sensitivity of the assay to detect endogenous pS421-HTT in cerebrospinal fluid (CSF) from nonhuman primates, showing for the first time that phosphorylation at S421-HTT can be detected in this bio-fluid. These reagents and assay will enable investigation of the biological consequence and the relevance of pS421 in the natural history of HD. ### Competing Interest Statement The authors have declared no competing interest.

Huntington’s disease (HD) is fatal neurodegenerative disorder caused by the expansion of a CAG-repeat tract in the huntingtin ( HTT ) gene. Human and mouse genetics studies have demonstrated a role for DNA mismatch repair (MMR) proteins which control the rate of somatic expansion of the HTT CAG repeat and disease onset and progression. MutSβ, a key member of the MMR pathway, is a heterodimeric protein of MSH2 and MSH3 that recognizes and initiates the repair of small insertion or deletion DNA loop outs. Both mouse Msh3 loss-of-function and reduced-expression alleles of human MSH3 lead to slower rates of somatic expansion in the HTT CAG tract and a delay of disease onset and progression, signifying MSH3 as a promising drug target for HD. Structural biology studies of MutSβ are informative for mechanism, protein structure-function relationships, and guiding small-molecule drug design. Here we report biochemical and cryo-electron microscopy analyses of human MutSβ ensembles, revealing that MutSβ undergoes multiple conformational changes in response to binding and release of nucleotides and DNA. The DNA-free MutSβ-ADP complex adopts an open conformation that is compatible with DNA binding. The conformation of MutSβ in the (CAG) 2 DNA-bound open structure most closely resembles the recently identified low-affinity state of MutSα, compared to the canonical mismatch-bound conformation. The homoduplex-bound and DNA-unbound MutSβ-ATP structures show that MutSβ undergoes an ATP-dependent conformational change towards sliding clamp forms. This study provides a comprehensive understanding of the structural conformational dynamics of MutSβ, insights into the MMR cascade, and a foundation for structure-guided drug discovery.

Background Evidence from animal studies and post-mortem studies of brains from people with Huntington's disease (PwHD) has suggested that the kynurenine pathway (KP) may be dysregulated in Huntington's disease (HD). Objective To determine whether there are differences in KP metabolites in the cerebrospinal fluid (CSF) and plasma of PwHD vs. healthy controls enrolled in the HDClarity study. Methods CSF and plasma samples from 141 PwHD with mild and moderate manifest disease and 75 healthy controls were analyzed for 3-hydroxykynurenine (3-OH-KYN), quinolinic acid, kynurenine, anthranilic acid, kynurenic acid, and tryptophan concentrations using validated high-performance liquid chromatography with tandem mass spectrometry methods. The primary and secondary endpoints compared metabolite concentrations between groups, and an exploratory analysis (PwHD only) evaluated the association between the metabolite levels and severity of disease. Results No significant differences in CSF or plasma concentrations of any of the six KP metabolites were observed between PwHD and controls, and there were no strong associations between the concentration of any KP metabolite and disease severity. A principal component analysis of the combined CSF and plasma measures showed a substantial positive correlation among all metabolites except for tryptophan in plasma. Conclusions We found no evidence to support the hypothesis of dysregulation of KP metabolites in HD based on CSF and plasma metabolite levels. The monitoring of KP metabolites in CSF or plasma is unlikely to serve as a pharmacodynamic biomarker for disease progression or therapeutic intervention in HD.

Positron emission tomography (PET) imaging of mutant huntingtin (mHTT) aggregates is a potential tool to monitor disease progression as well as the efficacy of candidate therapeutic interventions for Huntington’s disease (HD). To date, the focus has been mainly on the investigation of 11C radioligands; however, favourable 18F radiotracers will facilitate future clinical translation. This work aimed at characterising the novel [18F]CHDI-650 PET radiotracer using a combination of in vivo and in vitro approaches in a mouse model of HD. After characterising [18F]CHDI-650 using in vitro autoradiography, we assessed in vivo plasma and brain radiotracer stability as well as kinetics through dynamic PET imaging in the heterozygous (HET) zQ175DN mouse model of HD and wild-type (WT) littermates at 9 months of age. Additionally, we performed a head-to-head comparison study at 3 months with the previously published [11C]CHDI-180R radioligand. Plasma and brain radiometabolite profiles indicated a suitable metabolic profile for in vivo imaging of [18F]CHDI-650. Both in vitro autoradiography and in vivo [18F]CHDI-650 PET imaging at 9 months of age demonstrated a significant genotype effect (p < 0.0001) despite the poor test–retest reliability. [18F]CHDI-650 PET imaging at 3 months of age displayed higher differentiation between genotypes when compared to [11C]CHDI-180R. Overall, [18F]CHDI-650 allows for discrimination between HET and WT zQ175DN mice at 9 and 3 months of age. [18F]CHDI-650 represents the first suitable 18F radioligand to image mHTT aggregates in mice and its clinical evaluation is underway.

Orthosteric inhibitors of the human heterodimeric DNA mismatch repair complex MutSbeta were identified by high-throughput screening. Following extensive hit confirmation to remove false positives, two series were found to give consistent activity free of likely artefactual effects. Extensive hit profiling confirmed an ATP-competitive mode of action, and X-ray crystallography showed the inhibitors occupying the ATP-binding site of MSH3.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (CAG) trinucleotide expansion in the huntingtin (HTT) gene that encodes the mutant huntingtin protein (mHTT). Visualization and quantification of cerebral mHTT will provide a proxy for target engagement and a means to evaluate therapeutic interventions aimed at lowering mHTT in the brain. Here, we validated the novel radioligand C-11-labeled 6-(5-((5-methoxypyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one (C-11-CHDI-180R) using PET imaging to quantify cerebral mHTT aggregates in a macaque model of HD. Methods: Rhesus macaques received MRI-guided intrastriatal delivery of a mixture of AAV2 and AAV2.retro viral vectors expressing an HTT fragment bearing 85 CAG repeats (85Q, n = 5), a control HTT fragment bearing 10 CAG repeats (10Q, n = 4), or vector diluent only (phosphate-buffered saline, n = 5). Thirty months after surgery, 90-min dynamic PET/CT imaging was used to investigate C-11-CHDI-180R brain kinetics, along with serial blood sampling to measure input function and stability of the radioligand. The total volume of distribution was calculated using a 2-tissue-compartment model as well as Logan graphical analysis for regional quantification. Immunostaining for mHTT was performed to corroborate the in vivo findings. Results: C-11-CHDI-180R displayed good metabolic stability (51.4% +/- 4.0% parent in plasma at 60 min after injection). Regional time-activity curves displayed rapid uptake and reversible binding, which were described by a 2-tissue-compartment model. Logan graphical analysis was associated with the 2-tissue-compartment model (r(2) = 0.96, P < 0.0001) and used to generate parametric volume of distribution maps. Compared with controls, animals administered the 85Q fragment exhibited significantly increased C-11-CHDI-180R binding in several cortical and subcortical brain regions (group effect, P < 0.0001). No difference in C-11-CHDI-180R binding was observed between buffer and 10Q animals. The presence of mHTT aggregates in the 85Q animals was confirmed histologically. Conclusion: We validated C-11-CHDI-180R as a radioligand to visualize and quantify mHTT aggregated species in a HD macaque model. These findings corroborate our previous work in rodent HD models and show that C-11-CHDI-180R is a promising tool to assess the mHTT aggregate load and the efficacy of therapeutic strategies.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntington's disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner, dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical interferon regulatory factor3-mediated IKK pathway, distinct from the established involvement of IKBKB in mutant huntingtin's pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntington's disease.

Huntington’s disease is caused by a trinucleotide expansion in the HTT gene, which leads to aggregation of mutant huntingtin (mHTT) protein in the brain and neurotoxicity. Direct in vivo measurement of mHTT aggregates in human brain parenchyma is not yet possible. In this first-in-human study, we investigated biodistribution and dosimetry in healthy volunteers of [11C]CHDI-00485180-R ([11C]CHDI-180R) and [11C]CHDI-00485626 ([11C]CHDI-626), two tracers designed for PET imaging of aggregated mHTT in the brain that have been validated in preclinical models. Biodistribution and radiation dosimetry studies were performed in 3 healthy volunteers (age 25.7 ± 0.5 years; 2 F) for [11C]CHDI-180R and in 3 healthy volunteers (age 35.3 ± 6.8 years; 2 F) for [11C]CHDI-626 using sequential whole-body PET-CT. Source organs were delineated in 3D using combined PET and CT data. Individual organ doses and effective doses were determined using OLINDA 2.1. There were no clinically relevant adverse events. The mean effective dose (ED) for [11C]CHDI-180R was 4.58 ± 0.65 μSv/MBq, with highest absorbed doses for liver (16.9 μGy/MBq), heart wall (15.9 μGy/MBq) and small intestine (15.8 μGy/MBq). Mean ED for [11C]CHDI-626 was 5.09 ± 0.06 μSv/MBq with the highest absorbed doses for the gallbladder (26.5 μGy/MBq), small intestine (20.4 μGy/MBq) and liver (19.6 μGy/MBq). Decay-corrected brain uptake curves showed promising kinetics for [11C]CHDI-180R, but for [11C]CHDI-626 an increasing signal over time was found, probably due to accumulation of a brain-penetrant metabolite. [11C]CHDI-180R and [11C]CHDI-626 are safe for in vivo PET imaging in humans. The estimated radiation burden is in line with most 11C-ligands. While [11C]CHDI-180R has promising kinetic properties in the brain, [11C]CHDI-626 is not suitable for human in vivo mHTT PET due to the possibility of a radiometabolite accumulating in brain parenchyma. EudraCT number 2020-002129-27. Clinicaltrials.gov NCT05224115 (retrospectively registered).

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin ( HTT ) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (polyQ) tract. Whereas several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, methods to visualize mHTT protein species in the living brain are lacking. Here, we demonstrate the development and characterization of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11 C ([ 11 C]CHDI-180R) allowed noninvasive monitoring of mHTT pathology in the brain and could track region- and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression in a rodent model. We further showed that in these animals, therapeutic agents that lowered mHTT in the striatum had a functional restorative effect that could be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.