ABSTRACT
The genetic mutation causing Huntington's disease is a polyglutamine expansion in the huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant huntingtin, understanding huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Förster resonance energy transfer (FRET) assay for quantification of soluble mutant huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant huntingtin. The design of the homogeneous one-step method for huntingtin detection is such that it can be easily applied to measure other proteins of interest.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Huntington Disease/diagnosis , Mutant Proteins/isolation & purification , Nerve Tissue Proteins/isolation & purification , Nuclear Proteins/isolation & purification , Adult , Analysis of Variance , Animals , Brain/metabolism , Cell Line , Disease Progression , Embryonic Stem Cells/metabolism , Exons , Female , Gene Expression , Humans , Huntingtin Protein , Huntington Disease/blood , Huntington Disease/cerebrospinal fluid , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Middle Aged , Muscle, Skeletal/metabolism , Mutant Proteins/metabolism , Neocortex/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Recombinant Fusion Proteins , Sensitivity and Specificity , Time Factors , Young AdultABSTRACT
The quantification of cellular proteins is essential for the study of many different biological processes. This study describes an assay for the detection of the intracellular mutant huntingtin, the causative agent of Huntington's disease, with a method that may be generally applicable to other cellular proteins. A small recombinant protein tag that is recognized by a pair of readily available, high-affinity monoclonal antibodies was designed. This tag was then added to an inducible fragment of the mutant huntingtin protein by genetic engineering. We show that it is possible to use time-resolved FRET to detect low intracellular levels of huntingtin by a simple lysis and detection procedure. This assay was then adapted into a homogeneous, miniaturized format suitable for screening in 1536-well plates. The use of time-resolved FRET also permits the assay to be multiplexed with a standard readout of cell toxicity, thus allowing the identification of conditions causing reduction of protein levels simply due to cytotoxicity. The screening results demonstrated that the assay is able to identify compounds that modulate the levels of huntingtin both positively and negatively and that represent valuable starting points for drug discovery programs.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Nerve Tissue Proteins/analysis , Nuclear Proteins/analysis , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Cell Line , Huntingtin Protein , Mice , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Oxazines/chemistry , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Small Molecule Libraries , Xanthenes/chemistryABSTRACT
Neurodegenerative diseases such as Huntington's, Parkinson's and Alzheimer's diseases are marked by neuronal accumulation of toxic misfolded protein. Developing therapies for these misfolding diseases requires finding chemical compounds that can either clear toxic misfolded protein, or can protect neurons from their impact. Such compounds could not only provide the starting points for potential drugs, but could also provide valuable research tools for untangling the complexities of the disease process. Until now, chemical screens for these diseases have focused on finding compounds that prevent aggregation of mutant protein. We recently published a compound, B2, which promotes the formation of large inclusions by mutant Huntingtin and alpha-synuclein, while rescuing some of the toxic effects of these proteins. As inclusions were long believed to be toxic to cells, this contradicts previous therapeutic approaches. At the same time, the results support growing evidence for the protective effects of inclusions. In this review, we discuss these results, and place them in the context of ongoing therapeutic discovery efforts for Huntington's disease and other neurodegenerative diseases.
Subject(s)
Inclusion Bodies/drug effects , Mutant Proteins/chemistry , Neurodegenerative Diseases/drug therapy , Humans , Neurodegenerative Diseases/etiology , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Nitroquinolines/therapeutic use , alpha-Synuclein/drug effectsABSTRACT
Misfolded proteins accumulate in many neurodegenerative diseases, including huntingtin in Huntington's disease and alpha-synuclein in Parkinson's disease. The disease-causing proteins can take various conformations and are prone to aggregate and form larger cytoplasmic or nuclear inclusions. One approach to the development of therapeutic intervention for these diseases has been to identify chemical compounds that reduce the size or number of inclusions. We have, however, identified a compound that promotes inclusion formation in cellular models of both Huntington's disease and Parkinson's disease. Of particular interest, this compound prevents huntingtin-mediated proteasome dysfunction and reduces alpha-synuclein-mediated toxicity. These results demonstrate that compounds that increase inclusion formation may actually lessen cellular pathology in both Huntington's and Parkinson's diseases, suggesting a therapeutic approach for neurodegenerative diseases caused by protein misfolding.
