New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins (2023)

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  • Cited by (148)
  • Recommended articles (6)

Brain Research Bulletin

Volume 56, Issues 3–4,

1 November 2001

, Pages 319-329

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We produced eight anti-huntingtin (Htt) monoclonal antibodies (mAbs), several of which have novel binding patterns. Peptide array epitope mapping shows that mAbs MW1–6 specifically bind the polyQ domain of Htt exon 1. On Western blots of extracts from mutant Htt knock-in mouse brain and Huntington’s disease lymphoblastoma cell lines, MW1-5 all strongly prefer to bind to the expanded polyQ repeat form of Htt, displaying no detectable binding to normal Htt. These results suggest that the polyQ domain can assume different conformations that are distinguishable by mAbs. This idea is supported by immunohistochemistry with wild type (WT) and mutant Htt transgenic mouse (R6) brains. Despite sharing the same epitope and binding preferences on Western blots, MW1–5 display distinct staining patterns. MW1 shows punctate cytoplasmic and neuropil staining, while MW2–5 strongly stain the neuronal Golgi complex. MW6, in contrast, stains neuronal somas and neuropil. In addition, despite their preference for mutant Htt on blots, none of these mAbs show enhanced staining of R6 brains over WT, and show no binding of the Htt-containing nuclear inclusions in R6 brains. This suggests that in its various subcellular locations, the polyQ domain of Htt either takes on different conformations and/or is differentially occluded by Htt binding proteins. In contrast to MW1–6, MW7, and 8 can differentiate transgenic from WT mice by staining nuclear inclusions in R6/2 brain; MW8 displays no detectable staining in WT brain and stains only inclusions in R6/2 brain. Epitope mapping reveals that MW7 and 8 specifically bind the polyP domains and amino acids 83-90, respectively. As with MW1-6, the epitopes for MW7 and 8 are differentially available in the various subcellular compartments where Htt is found.


Huntington’s disease (HD) is caused by the extension of a polyglutamine (polyQ) tract in the protein huntingtin (Htt) to a length >40 units [20]. Immunohistochemistry and subcellular fractionation indicate that Htt is normally located in the cytoplasm while the mutant form of Htt is also found in aggregates (inclusions) in the nucleus [4]. While this correlation with the disease suggests that translocation and/or aggregation in the nucleus is important for the neuronal cell death that occurs in HD, the importance of the inclusions themselves remains controversial [13]. A related question concerns the binding partners for Htt in the nucleus and the cytoplasm. Studies in yeast, Drosophila and mammalian cells have shown that various chaperone proteins can alter nuclear inclusion formation and/or the toxicity associated with expanded polyQ repeat Htt (as well as ataxin-3, another protein that causes neurodegenerative disease when its polyQ tract is extended) [4]. Other proteins also bind Htt and ataxin-3, which can lead to alterations in transcription 1, 12, 25. Regarding its normal function, Htt has been localized to various subcellular sites, including the neuronal cytoplasm, nucleus, presynaptic vesicles, varicosities, Golgi network, dendritic plasma membranes, and cytoskeleton [20]. Although it is not yet clear how much of this diversity in reported localization is genuine, it would not be surprising if a protein of >3,000 amino acids had multiple functions and sites of action [8], as well as many different binding partners in various locations.

Critical tools in many of such studies are anti-Htt antibodies, which can be used for immunohistochemistry, immunoprecipitation, structural studies, drug screening, and functional perturbation. Seeking to broaden the range of available anti-Htt monoclonal antibodies (mAbs), and to clarify the subcellular localization of Htt, we have produced eight new mAbs. We used as antigens several expanded polyQ constructs, including soluble Htt exon 1, as well as aggregates of Htt. Some of these mAbs display distinct immunohistochemical staining patterns not seen in prior studies. Coupled with Western blotting results, these staining patterns suggest that the conformation and/or the binding partners of Htt are different in various subcellular compartments and when it forms nuclear inclusions.

Section snippets

Production of mAbs

Six-week-old Balb/c female mice were primed and boosted at 2 week intervals by intraperitoneal injection of antigen emulsified in adjuvant (RIBI Immunochem, Hamilton, MT, USA). Test bleeds were obtained 7 days after every other injection. A final series of boosts was performed without adjuvant. Spleen cells were isolated from the mouse 3 days after the final boost and fused with HL-1 murine myeloma cells (Ventrex, Portland, ME, USA) using polyethylene glycol (PEG 1500, Boehringer-Mannheim,

Generation of the mAbs

For the first round of immunizations, we injected proteins expressed from two constructs containing the polyQ domain (19 or 35 repeats) and 34 amino acids of the dentatorubralpalliodoluysian atrophy (DRPLA) gene fused to glutathione-S-transferase (GST) [16]. Using enzyme linked substrate assay (ELISA) to screen against these antigens versus GST alone, we selected three hybridomas for cloning. These mAbs are termed MW (for Milton Wexler) 1, 2, and 5. As described below, while each of these mAbs


We thank James Burke, Marie-Francoise Chesselet, Vivian Hook, George Jackson, Parsa Kazemi-Esfarjani, Alex Kanzantsev, Ali Khoshnan, George Lawless, Marcy MacDonald, Hemachandro Reddy, Allan Sharp, Gabriele Shilling, Peter Snow, Leslie Thompson, Peter Thumfort, Jonathon Wood, and Scott Zeitlin for generously providing antibodies, cell lines, mouse tissues, and DNA constructs. Peter Snow expressed proteins and Peter Thumfort generously provided the peptide arrays and advice on epitope mapping.

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  • Cited by (148)

    • Molecular basis of Q-length selectivity for the MW1 antibody–huntingtin interaction

      2023, Journal of Biological Chemistry

      Huntington’s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1–MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600–2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

    • N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease

      2022, Neurobiology of Disease

      Citation Excerpt :

      MAB5492+ aggregate number did not correlate with CAG repeat length, symptom onset, SMI-32+ pyramidal cell loss or 1C2+ aggregates (Fig. 8 HK). MW8 was generated against human Htt exon 1 containing a 67-residue polyQ tract, resulting in its specificity for Htt N-terminal amino acids 83–90 (Fig. 9 A) (Ko et al., 2001). Qualitative examination of MW8 immunoreactivity demonstrated diffuse immunoreactivity localized to cell bodies in both HD and control tissue (Fig. 9 BC).

      Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs).

      Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5–12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

    • Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells

      2022, SLAS Discovery

      Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.

    • Structural Model of the Proline-Rich Domain of Huntingtin Exon-1 Fibrils

      2020, Biophysical Journal

      Huntington’s disease is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine domain that increases the propensity of huntingtin exon-1 to form cross-β fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800-ns long molecular dynamics simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II helix for most of the simulation, whereas occasional kinks in the proline-rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.

    • Optimizing intracellular antibodies (intrabodies/nanobodies) to treat neurodegenerative disorders

      2020, Neurobiology of Disease

      Intrabodies (both single-chain Fv and single-domain VH, VHH, and VL nanobodies) offer unique solutions to some of the challenges of delivery and target engagement posed by immunotherapeutics for the brain and other areas of the nervous system. The specificity, which includes the recognition of post-translational modifications, and capacity for engineering that characterize these antibody fragments can be especially well-focused when the genes encoding only the binding sites of the antibody are expressed intracellularly. Multifunctional constructs use fusions with peptides that can re-target antigen-antibody complexes to enhance both pharmacodynamic activity and intracellular solubility simultaneously. Fusions with proteolytic targeting signals, such as the PEST degron, greatly enhance potency in some cases. Stem cell transplants can be protected from exogenous misfolded proteins by stable transfection with intrabodies. Tandem expression to target two or more misfolding proteins in one treatment may be especially valuable for proteostatic disruptions due to genetic, aging, or toxic triggers. Advances in bioinformatics, screening protocols, and especially gene therapy are showing great promise for intrabody/ nanobody treatments of a full range of neurological disorders, including Alzheimer's disease and related tau dementias, Parkinson's disease and Lewy body diseases, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases, among others.

    • Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD

      2020, Neuropharmacology

      One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds – the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

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