Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay.

BACKGROUND: The use of biomarkers has become a major component of clinical trial design. In Huntington's disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein. OBJECTIVE: We aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples. METHODS: An ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples. RESULTS: A novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF. CONCLUSION: We have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

Analysis of mutant and total huntingtin expression in Huntington's disease murine models.

Huntington's disease (HD) is a monogenetic neurodegenerative disorder that is caused by the expansion of a polyglutamine region within the huntingtin (HTT) protein, but there is still an incomplete understanding of the molecular mechanisms that drive pathology. Expression of the mutant form of HTT is a key aspect of diseased tissues, and the most promising therapeutic approaches aim to lower expanded HTT levels. Consequently, the investigation of HTT expression in time and in multiple tissues, with assays that accurately quantify expanded and non-expanded HTT, are required to delineate HTT homeostasis and to best design and interpret pharmacodynamic readouts for HTT lowering therapeutics. Here we evaluate mutant polyglutamine-expanded (mHTT) and polyglutamine-independent HTT specific immunoassays for validation in human HD and control fibroblasts and use to elucidate the CSF/brain and peripheral tissue expression of HTT in preclinical HD models.

Quantifying autophagy using novel LC3B and p62 TR-FRET assays.

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay.

BACKGROUND: The measurement of disease-relevant biomarkers has become a major component of clinical trial design, but in the absence of rigorous clinical and analytical validation of detection methodology, interpretation of results may be misleading. In Huntington's disease (HD), measurement of the concentration of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technology and here analytically validate it for use. OBJECTIVE: This work aims to analytically and clinically validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clinical trials. METHODS: The single molecule counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technology was clinically validated following established Food and Drug Administration and European Medicine Agency guidelines. RESULTS: The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addition, a set of recommendations on result interpretation is provided. CONCLUSIONS: This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.

Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models.

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.

Recombinant expression and insecticidal properties of a Conus ventricosus conotoxin-GST fusion protein.

A novel conotoxin, conotoxin Vn2, was recently isolated from the venom of Conus ventricosus, a worm-hunting cone snail species living in the Mediterranean Sea. Analysis of conotoxin Vn2 amino acid sequence suggested that it is a member of the O1 superfamily of conotoxins. Conotoxin Vn2 displays quite a high degree of sequence similarity with bioactive peptides targeting calcium channels and in particular with the ω conotoxin PnVIB, extracted from the venom of the molluscivorous cone snail Conus pennaceus. In this work we describe the development of a heterologous expression system to obtain a glutathione-S-transferase (GST) fusion product of conotoxin Vn2 in a pure form and in a sufficient amount to characterize its bioactivity. The fusion product has been expressed in recombinant form in Escherichia coli cells, purified, and its neurotoxic activity has been assayed on the larvae of the moth Galleria mellonella, a simple experimental model to test the toxicity of compounds in insects. Moreover the conotoxin Vn2 Asp2His mutant has been produced to analyse the role of this aspartic acid residue in the toxin bioactivity, as an acidic amino acid is conserved in this position in all the O1 superfamily C. ventricosus conotoxins. Results obtained indicate that indeed conotoxin Vn2 has strong insecticidal properties at a dose of only 100 pmol/g of body weight. Surprisingly, mutation of Asp2 to His leads to enhanced toxicity in the larvae model system opening up interesting possibilities for the use of conotoxin Vn2 variants in environmental friendly crop protection applications.

Cupricyclins, novel redox-active metallopeptides based on conotoxins scaffold.

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu(2+) ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins.