IKBKB reduces huntingtin aggregation by phosphorylating serine 13 via a non-canonical IKK pathway.

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntington's disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner, dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical interferon regulatory factor3-mediated IKK pathway, distinct from the established involvement of IKBKB in mutant huntingtin's pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntington's disease.

Striatal infusion of cholesterol promotes dose-dependent behavioral benefits and exerts disease-modifying effects in Huntington's disease mice.

A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.

Phospho-S129 Alpha-Synuclein Is Present in Human Plasma but Not in Cerebrospinal Fluid as Determined by an Ultrasensitive Immunoassay.

Accumulation and aggregation of misfolded alpha-synuclein is believed to be a cause of Parkinson's disease (PD). Phosphorylation of alpha-synuclein at S129 is known to be associated with the pathological misfolding process, but efforts to investigate the relevance of this post-translational modification for pathology have been frustrated by difficulties in detecting and quantifying it in relevant samples. We report novel, ultrasensitive immunoassays based on single-molecule counting technology, useful for detecting alpha-synuclein and its S129 phosphorylated form in clinical samples in the low pg/ml range. Using human CSF and plasma samples, we find levels of alpha-synuclein comparable to those previously reported. However, while alpha-synuclein phosphorylated on S129 could easily be detected in human plasma, where its detection is extremely sensitive to protein phosphatases, its levels in CSF were undetectable, with a possible influence of a matrix effect. In plasma samples from a small test cohort comprising 5 PD individuals and five age-matched control individuals we find that pS129 alpha-synuclein levels are increased in PD plasma samples, in line with previous reports. We conclude that pS129 alpha-synuclein is not detectable in CSF and recommend the addition of phosphatase inhibitors to plasma samples at the time of collection. Moreover, the findings obtained on the small cohort of clinical plasma samples point to plasma pS129 alpha-synuclein levels as a candidate diagnostic biomarker in PD.

Inhibiting pathologically active ADAM10 rescues synaptic and cognitive decline in Huntington's disease.

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.

Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease.

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.

Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.

Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKß.

Expansion of a CAG triplet repeat within the first exon of the HUNTINGTIN gene encoding for a polyglutamine tract is the cause of a progressive neurodegenerative disorder known as Huntington's disease. N-terminal fragments of mutant huntingtin have a strong propensity to form oligomers and aggregates that have been linked to the Huntington's disease pathology by different mechanisms, including gain of toxic functions. The biological and biophysical properties of the polyglutamine expansion within these huntingtin fragments are influenced by neighboring domains, in particular by the first 17 amino acids of huntingtin (N17), which precede the polyglutamine expansion. It has been suggested that N17 phosphorylation modulate mutant huntingtin aggregation and toxicity, but the study of its functional and pathological relevance requires the capacity to detect this modification in biological samples in a simple, robust way, that ideally provides information on the abundance of a phosphorylated species relative to the total pool of the protein of interest. Using a modified SDS-PAGE protocol (Phos-Tag) followed by Western blotting with specific anti-HUNTINGTIN antibodies, we efficiently resolved huntingtin fragments expressed in cellular contexts based on the presence of phosphorylated residues, we defined threonine 3 as the major site of huntingtin N17 phosphorylation and, finally, we identified IKK-beta as a kinase capable of phosphorylating threonine 3 in N-terminal hungtingtin fragments.

Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

BACKGROUND: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. METHODOLOGY/PRINCIPAL FINDINGS: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. CONCLUSIONS/SIGNIFICANCE: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Identification and characterization of a small-molecule inhibitor of Wnt signaling in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.

Solubilization methods and reference 2-DE map of cow milk fat globules.

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.