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

doi:10.1016/S0361-9230(01)00599-8

Brain Research Bulletin

Volume 56, Issues 3–4, 1 November 2001, Pages 319-329

https://doi.org/10.1016/S0361-9230(01)00599-8 Get rights and content

Abstract

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.

Introduction

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

Acknowledgements

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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Expansion of a triplet repeat tract in exon 1 of the HTT gene causes Huntington’s disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. Lowering mHTT is a promising approach to treat HD, but it is unclear when lowering should be initiated, how much is necessary, and what duration should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model, we analyzed mHtt lowering initiated at different ages and sustained for different time-periods. mHTT protein in cytoplasmic and synaptic compartments of the striatum was reduced 38-52%; however, there was minimal lowering of mHTT in nuclear and perinuclear regions where aggregates formed at 12 months of age. Total striatal lipids were reduced in 9-month-old LacQ140 mice and preserved by mHtt lowering. Subclasses important for white matter structure and function including ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), contributed to the reduction in total lipids. Phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses except ceramide were preserved by mHtt lowering. mRNA expression profiling indicated that a transcriptional mechanism contributes to changes in myelin lipids, and some but not all changes can be prevented by mHtt lowering. Our findings suggest that early and sustained reduction in mHtt can prevent changes in levels of select striatal proteins and most lipids, but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.
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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
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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.
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New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins

Abstract

Introduction

Section snippets

Production of mAbs

Generation of the mAbs

Acknowledgements

Trends Neurosci.

Curr. Biol.

Cell

Neuron

Curr. Opin. Neurobiol.

J. Immunol. Methods

Neuron

Cell

Exp. Neurol.

FEBS Lett.

Trends Neurosci.

Neuron

Exp. Neurol.

Mol. Cell

Trends Cell. Biol.