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.

Recombinant Adeno Associated Viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches.

Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington's disease.

Although Huntington's disease is caused by the expansion of a CAG triplet repeat within the context of the 3144-amino acid huntingtin protein (HTT), studies reveal that N-terminal fragments of HTT containing the expanded PolyQ region can be produced by proteolytic processing and/or aberrant splicing. N-terminal HTT fragments are also prevalent in postmortem tissue, and expression of some of these fragments in model organisms can cause pathology. This has led to the hypothesis that N-terminal peptides may be critical modulators of disease pathology, raising the possibility that targeting aberrant splicing or proteolytic processing may present attractive therapeutic targets. However, many factors can contribute to pathology, including genetic background and differential expression of transgenes, in addition to intrinsic differences between fragments and their cellular effects. We have used Drosophila as a model system to determine the relative toxicities of different naturally occurring huntingtin fragments in a system in which genetic background, transgene expression levels and post-translational proteolytic processing can be controlled. These studies reveal that among the naturally occurring N-terminal HTT peptides, the exon 1 peptide is exceptionally pathogenic and exhibits unique structural and biophysical behaviors that do not appear to be incremental changes compared with other fragments. If this proves correct, efforts to specifically reduce the levels of exon 1 peptides or to target toxicity-influencing post-translational modifications that occur with the exon 1 context are likely to have the greatest impact on pathology.

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.

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease.

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

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.

A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction.

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

Identification of a candidate alternative promoter region of the human Bcl2L11 (Bim) gene.

BACKGROUND: Despite the importance of the BCL2L11 (BIM) protein in various apoptotic processes in development and disease, little is known of the promoter structure of the human BCL2L11 locus and of the cis-acting elements regulating expression of the human gene. RESULTS: In the search for novel promoter sequences in the human BCL2L11 locus, we have identified previously unrecognized genomic sequences displaying promoter activity and E2F responsiveness, and driving the expression of BCL2L11 coding transcripts. In man, transcripts originating from this novel putative promoter contribute significantly to total BCL2L11 mRNA expression in testis, heart and liver. In HEK293 cells, this novel candidate promoter originates BCL2L11 transcripts whose expression can be modulated by a known modulator of BCL2L11 expression (Trichostatin A) and by E2F, a characterized transcriptional regulator of BCL2L11 expression. CONCLUSION: The identification of a novel putative human BCL2L11 promoter provides new insights into the structure and regulation of the BCL2L11 locus.

Differential activation of the calcium/protein kinase C and the canonical beta-catenin pathway by Wnt1 and Wnt7a produces opposite effects on cell proliferation in PC12 cells.

We examined the effect of Wnt1 and Wnt7a on cell proliferation using undifferentiated PC12 cells, which originate from the neural crest and are widely employed as a neuronal cell model. Heterologous expression of Wnt1 enhanced [3H]thymidine incorporation and expression of cyclin D1 and cylin E in PC12 cells. Opposite effects were observed in PC12 cells expressing Wnt7a. Searching for the mechanisms underlying the opposite effects of Wnt1 and Wnt7a on PC12 cell proliferation, we examined the activation of the canonical beta-catenin/T-cell-lymphoid enhancer-binding protein transcription factor pathway and the 'calcium pathway' by co-transfecting the cells with a reporter gene controlled by either T-cell-lymphoid enhancer-binding protein transcription factor or the calcium-activated transcription factor, NFAT. Wnt1 and Wnt7a activated both pathways, but to a different extent. While Wnt1 preferentially activated the calcium pathway, Wnt7a mainly activated the canonical pathway. Pharmacological inhibition of protein kinase C, which is a component of the calcium pathway, abrogated the increase in cell proliferation induced by Wnt1 without affecting the antiproliferative action of Wnt7a. The action of Wnt7a was instead occluded by lithium ions, which mimic the activation of the canonical pathway, and was largely reduced by Dickkopf-1, which acts as an inhibitor of the canonical pathway. In addition, expression of a constitutively active mutant of beta-catenin potently activated the canonical Wnt pathway and reduced [3H]thymidine incorporation. These data challenge the view that the canonical Wnt pathway invariably supports cell growth and suggest that, at least in PC12 cells, cell proliferation is regulated by the balance between the calcium/protein kinase C pathway and the canonical pathway.

Inhibition of Wnt signaling, modulation of Tau phosphorylation and induction of neuronal cell death by DKK1.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimer's Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Inhibition of the canonical Wnt signaling pathway by apolipoprotein E4 in PC12 cells.

We examined the effect of the three human isoforms of apolipoprotein E (ApoE2, ApoE3, and ApoE4) on the canonical Wnt signaling pathway in undifferentiated PC12 cells. Addition of recombinant ApoE4 reduced Wingless-Int7a-stimulated gene expression at concentrations of 80 and 500 nm. Recombinant ApoE2 and ApoE3 were virtually inactive. Recombinant ApoE4 also inhibited Wnt signaling when combined with very low density lipoproteins (VLDLs) or in cells over-expressing the low density lipoprotein receptor-related protein, LRP6. In contrast, the enforced expression of LRP5 unmasked an inhibition by ApoE2 and ApoE3, which, however, were less effective than ApoE4 in inhibiting Wnt signaling. We also transfected PC12 cells with constructs encoding for the three human ApoE isoforms to examine whether endogenously expressed ApoE isoforms could modulate the Wnt pathway. Under these conditions, all three ApoE isoforms were able to inhibit Wnt signaling, although ApoE4 showed the greatest efficacy. Only the conditioned medium collected from cultures transfected with ApoE4 induced a significant inhibition of Wnt7a-stimulated gene expression, confirming that ApoE4 has an extracellular action that is not shared by the other ApoE isoforms. We conclude that ApoE4 behaves as an inhibitor of the canonical Wnt pathway in a context-independent manner.

Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain.

We used primary cultures of cortical neurons to examine the relationship between beta-amyloid toxicity and hyperphosphorylation of the tau protein, the biochemical substrate for neurofibrillary tangles of Alzheimer's brain. Exposure of the cultures to beta-amyloid peptide (betaAP) induced the expression of the secreted glycoprotein Dickkopf-1 (DKK1). DKK1 negatively modulates the canonical Wnt signaling pathway, thus activating the tau-phosphorylating enzyme glycogen synthase kinase-3beta. DKK1 was induced at late times after betaAP exposure, and its expression was dependent on the tumor suppressing protein p53. The antisense induced knock-down of DKK1 attenuated neuronal apoptosis but nearly abolished the increase in tau phosphorylation in betaAP-treated neurons. DKK1 was also expressed by degenerating neurons in the brain from Alzheimer's patients, where it colocalized with neurofibrillary tangles and distrophic neurites. We conclude that induction of DKK1 contributes to the pathological cascade triggered by beta-amyloid and is critically involved in the process of tau phosphorylation.

Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

The Wnt pathway, cell-cycle activation and beta-amyloid: novel therapeutic strategies in Alzheimer's disease?

Beta-amyloid protein (betaAP) is thought to cause neuronal loss in Alzheimer's disease (AD). Applied to neurons in culture, betaAP induces neuronal death and hyperphosphorylation of tau protein, which forms neurofibrillary tangles (NFTs) in AD brains. Neurons also undergo rapid apoptotic death following reactivation of a mitotic cycle. However, the molecular events that determine the fate of neurons challenged with betaAP (apoptotic death, formation of NFTs and survival) are unclear. We discuss a scenario for the pathogenesis of AD. This links betaAP-induced changes to the Wnt signaling pathway that promotes proliferation of progenitor cells and directs cells into a neuronal phenotype during brain development. We propose that betaAP-mediated facilitation of mitogenic Wnt signaling activates unscheduled mitosis in differentiated neurons. Furthermore, late downregulation of Wnt signaling by betaAP might lead to NFT formation. We propose that drugs that both inhibit the cell cycle and rescue Wnt activity could provide novel AD therapeutics.

Molecular cloning and initial characterization of the MG61/PORC gene, the human homologue of the Drosophila segment polarity gene Porcupine.

Insect and vertebrate Porcupine genes encode multi-pass endoplasmic reticulum proteins involved in the processing of Wnt (wingless and int homologue) proteins, a class of secreted glycoprotein factors homologous to the Drosophila melanogaster segment polarity gene Wingless (Wg). Here we report the cloning of cDNAs encoding the human homologue of the Drosophila gene Porcupine (Porc), the characterization of its genomic structure and the quantitative analysis of its expression in a comprehensive panel of human tissues. The human Porcupine locus (MG61/PORC) spans 15 exons over approximately 12 kb of genomic sequence on Xp11.23. Real-time quantitative expression analysis reveals that MG61/PORC transcripts are expressed in multiple tissues, but are particularly abundant in the brain. Like its mouse and Xenopus homologues, MG61/PORC encodes four protein isoforms (A-D) generated through alternative splicing and expressed in a tissue-specific fashion. Finally, we present evidence indicating that MG61/PORC can influence the activity of a human Wnt7A expression construct in a T-cell factor-responsive reporter assay.