Intrabodies Binding the Proline-Rich Domains of Mutant Huntingtin Increase Its Turnover and Reduce Neurotoxicity

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 10/2008; 28(36):9013-20. DOI: 10.1523/JNEUROSCI.2747-08.2008
Source: PubMed


Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

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Available from: Amber Southwell, Dec 22, 2013
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    • "Hence, new strategies that specifically target Huntingtin regions flanking the polyQ have been tested. C4 and VL12.3 intrabodies, designed against the N17 region to inhibit nucleation and aggregation, showed encouraging effects in cell model [48,49], but only moderate improvements of R6/2 mice [12]. Hence, the performances of NP42T reported here in R6/2 mice should be greater in other HD models. "
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    ABSTRACT: Background In Huntington¿s disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice.ResultsTo this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections.Conclusions These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.
    Full-text · Article · Aug 2014
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    • "The involvement of PRR in the pathological process of HD can be observed from the fact that MW7 single-chain variable fragment, a monoclonal antibody recognizing Htt PRR, significantly inhibits the aggregation, as well as the neurotoxicity, induced by mutant Htt protein (Khoshnan et al., 2002; Southwell et al., 2008). Many tryptophan (WW) or Src homology 3 (SH3) domain-containing proteins have been identified that interact with the PRR of Htt, such as Htt yeast two-hybrid protein HYPA (also named FBP11) and HYPB (known as SETD2) (Faber et al., 1998), SH3GL3/endophilin-A3 (Sittler et al., 1998), protein kinase C and casein kinase substrate in neuron 1 (PACSIN1/ syndapin) (Modregger et al., 2002), and CA150 (Holbert et al., 2001). "
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    ABSTRACT: Huntington's disease (HD) is an autosomally dominant neurodegenerative disorder caused by expansion of polyglutamine (polyQ) in the huntingtin (Htt) protein. Htt yeast two-hybrid protein B (HYPB/SETD2), a histone methyltransferase, directly interacts with Htt and is involved in HD pathology. Using NMR techniques, we characterized a polyproline (polyP) stretch at the C terminus of HYPB, which directly interacts with the following WW domain and leads this domain predominantly to be in a closed conformational state. The solution structure shows that the polyP stretch extends from the back and binds to the WW core domain in a typical binding mode. This autoinhibitory structure regulates interaction between the WW domain of HYPB and the proline-rich region (PRR) of Htt, as evidenced by NMR and immunofluorescence techniques. This work provides structural and mechanistic insights into the intramolecular regulation of the WW domain in Htt-interacting partners and will be helpful for understanding the pathology of HD.
    Full-text · Article · Jan 2014 · Structure
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    • "Most viral vectors allow an efficient integration of a gene of interest in the genome of dividing and non-dividing cells, which facilitates the generation of stable cell lines expressing intrabodies in suitable amounts [59] [60] [61]. Viral vectors were successfully applied to transduce neurons [62], model cell lines or xenograft models [63] with target specific intrabodies for different functional studies [62] [63] [64] [65] [66]. 4.2. "
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    ABSTRACT: In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Beside their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
    Full-text · Article · Jan 2014 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
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