Disruption of the nuclear membrane by perinuclear inclusions of mutant huntingtin causes cell-cycle re-entry and striatal cell death in mouse and cell models of Huntington's disease.

Accumulation of N-terminal fragments of mutant huntingtin (mHTT) in the cytoplasm, nuclei and axons of neurons is a hallmark of Huntington's disease (HD), although how these fragments negatively impact neurons remains unclear. We followed the distribution of mHTT in the striata of transgenic R6/2-J2 HD mice as their motor function declined. The fraction of cells with diffuse, perinuclear or intranuclear mHTT changed in parallel with decreasing motor function. In transgenic mice, medium spiny neurons (MSNs) that exhibited perinuclear inclusions expressed cell-cycle markers typically not seen in the striata of normal mice, and these cells are preferentially lost as disease progresses. Electron microscopy reveals that perinuclear inclusions disrupt the nuclear envelope. The progression of perinuclear inclusions being accompanied by cell-cycle activation and culminating in cell death was also observed in 1° cortical neurons. These observations provide a strong correlation between the subcellular location of mHTT, disruption of the nucleus, re-entry into the cell-cycle and eventual neuronal death. They also highlight the fact that the subcellular distribution of mHTT is highly dynamic such that the distribution of mHTT observed depends greatly on the stage of the disease being examined.

Mithramycin is a gene-selective Sp1 inhibitor that identifies a biological intersection between cancer and neurodegeneration.

Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington's disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21(waf1/cip1)) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.

Pcaf modulates polyglutamine pathology in a Drosophila model of Huntington's disease.

Huntingtin peptides with elongated polyglutamine domains, the root causes of Huntington's disease, hinder histone acetylation, which leads to transcriptional dysregulation. However, the range of acetyltransferases interacting with mutant Huntingtin has not been systematically evaluated. We used genetic interaction tests in Drosophila to determine whether specific acetyltransferases belonging to distinct protein families influence polyglutamine pathology. We found that flies expressing a mutant form of the Huntingtin protein (Httex1pQ93) exhibit reduced viability, which is further decreased by partial loss of Pcaf or nejire, while the tested MYST family acetyltransferases did not affect pathology. Reduced levels of Pcaf also led to the increased degeneration of photoreceptor neurons in the retina. Overexpression of Pcaf, however, was not sufficient to ameliorate these phenotypes, and the level of soluble Pcaf is unchanged in Httex1pQ93-expressing flies. Thus, our results indicate that while Pcaf has a significant impact on Huntington's disease pathology, therapeutic strategies aimed at elevating its levels are likely to be ineffective in ameliorating Huntington's disease pathology; however, strategies that aim to increase the specific activity of Pcaf remain to be tested.

A novel interaction between fibroblast growth factor receptor 3 and the p85 subunit of phosphoinositide 3-kinase: activation-dependent regulation of ERK by p85 in multiple myeloma cells.

Ectopic activation of fibroblast growth factor receptor 3 (FGFR3) is associated with several cancers, including multiple myeloma (MM). FGFR3 inhibition in these cells inhibits proliferation and induces apoptosis, validating FGFR3 signaling as a therapeutic target in t(4;14) MM cases. We have identified the PI3K regulatory subunit, p85alpha, as a novel interactor of FGFR3 by yeast two-hybrid, and confirmed an interaction with both p85alpha and p85beta in mammalian cells. The interaction of FGFR3 with p85 is dependent upon receptor activation. In contrast to the Gab1-mediated association of FGFRs with p85, the FGFR3-p85 interaction we observed requires FGFR3 Y760, previously identified as a PLCgamma binding site. The interaction of p85 with FGFR3 does not require PLCgamma, suggesting the p85 interaction is direct and independent of PLCgamma binding. FGFR3 and p85 proteins also interact in MM cell lines which consistently express p85alpha and p85beta, but not p50 or p55 subunits. siRNA knockdown of p85beta in MM cells caused an increased ERK response to FGF2. These data suggest that an endogenous negative regulatory role for the p85-FGFR3 interaction on the Ras/ERK/MAPK pathway may exist in response to FGFR3 activity and identifies a novel therapeutic target for MM.

Drosophila in the study of neurodegenerative disease.

As populations benefit from increasing lifespans, neurodegenerative diseases have emerged as a critical health concern. How can the fruit fly, Drosophila melanogaster, contribute to curing human diseases of the nervous system? A growing number of neurodegenerative diseases, as well as other human diseases, are being modeled in Drosophila and used as a platform to identify and validate cellular pathways that contribute to neurodegeneration and to identify promising therapeutic targets by using a variety of approaches from screens to target validation. The unique properties and tools available in the Drosophila system, coupled with the fact that testing in vivo has proven highly productive, have accelerated the progress of testing therapeutic strategies in mice and, ultimately, humans. This review highlights selected recent applications to illustrate the use of Drosophila in studying neurodegenerative diseases.

Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity.

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.

Inhibition of specific HDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is associated with transcriptional dysregulation, and multiple studies with histone deacetylase (HDAC) inhibitors suggest that global approaches for restoring transcriptional balance and appropriate protein acetylation are therapeutically promising. To determine whether more targeted approaches might be effective, we have tested the impact of all the HDACs in Drosophila on Huntingtin (Htt)-induced pathology. Among the zinc-dependent or 'classic' HDACs, we find that neurodegeneration is most sensitive to levels of Rpd3. We also find that among the NAD(+)-dependent class III deacetylases, genetic or pharmacological reduction of either Sir2 or Sirt2 provides neuroprotection to Htt-challenged animals and that even greater neuroprotection is achieved when Rpd3 and Sir2 are simultaneously reduced. Our experiments suggest that longevity promoting strategies may be distinct from those that protect against neurodegeneration in Drosophila challenged with mutant human Htt. These results highlight a novel therapeutic approach for HD in the form of Sir2 inhibition and possible combinatorial inhibition of Sir2 and Rpd3.

Nicotinamide restores cognition in Alzheimer's disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of Thr231-phosphotau.

Memory loss is the signature feature of Alzheimer's disease, and therapies that prevent or delay its onset are urgently needed. Effective preventive strategies likely offer the greatest and most widespread benefits. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance memory and synaptic plasticity. We evaluated the efficacy of nicotinamide, a competitive inhibitor of the sirtuins or class III NAD(+)-dependent HDACs in 3xTg-AD mice, and found that it restored cognitive deficits associated with pathology. Nicotinamide selectively reduces a specific phospho-species of tau (Thr231) that is associated with microtubule depolymerization, in a manner similar to inhibition of SirT1. Nicotinamide also dramatically increased acetylated alpha-tubulin, a primary substrate of SirT2, and MAP2c, both of which are linked to increased microtubule stability. Reduced phosphoThr231-tau was related to a reduction of monoubiquitin-conjugated tau, suggesting that this posttranslationally modified form of tau may be rapidly degraded. Overexpression of a Thr231-phospho-mimic tau in vitro increased clearance and decreased accumulation of tau compared with wild-type tau. These preclinical findings suggest that oral nicotinamide may represent a safe treatment for AD and other tauopathies, and that phosphorylation of tau at Thr231 may regulate tau stability.

CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.

Blood level of brain-derived neurotrophic factor mRNA is progressively reduced in rodent models of Huntington's disease: restoration by the neuroprotective compound CEP-1347.

Huntington's disease (HD) is an age-related neurodegenerative disorder that is currently untreatable. A prominent feature of HD pathology is the reduction of the pro-survival neurotrophin Brain-Derived Neurotrophic Factor (BDNF). Both mRNA and protein levels of BDNF are decreased in the brains of several HD rodent models and in human HD patients. We now report for the first time that this molecular event is mirrored in blood from HD rodent models. While protein levels of BDNF are undetectable in mouse blood, mRNA levels are measurable and diminish during HD progression in transgenic mouse (R6/2) and rat models of HD. Among the eight different BDNF transcripts, only BDNF exon III is transcribed in mouse blood and its expression is progressively compromised in R6/2 mice with respect to age-matched wild-types. Assessment of BDNF mRNA in HD rat blood shows a similar result, which is reinforced by evidence that protein levels of the neurotrophin are also significantly reduced at a symptomatic stage. Finally, we demonstrate that acute and chronic treatment of R6/2 mice with CEP-1347, a mixed lineage kinase (MLK) inhibitor with neuroprotective and neurotrophic effects, leads to increased total BDNF mRNA in blood when compared to untreated R6/2 mice. Our results indicate that alterations in BDNF mRNA levels in peripheral blood are a readily accessible measurement of disease progression and drug efficacy in HD rodent models.

A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.

Genes on distal chromosome 18 determine vulnerability to excitotoxic neurodegeneration following status epilepticus, but not striatal neurodegeneration induced by quinolinic acid.

Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part of chromosome 18 (chr18) is a major determinant of vulnerability to hippocampal neurodegeneration following kainic acid (KA)-induced seizures in inbred mouse strains. We assessed excitotoxic vulnerability in two congenic, "genome tagged" mouse strains carrying segments of either distal or proximal/medial chr18 from vulnerable DBA/2J mice on a resistant C57BL/6 background. Systemic KA injections triggered brain-wide neurodegeneration in the distal chr18 congenic strain, and specifically in the hilus of the dentate gyrus, but not in CA3. In contrast, the proximal/medial chr18 congenic strain exhibited enhanced degeneration in CA1 and CA3, but little neurodegeneration elsewhere. Both strains exhibited low levels of QUIN-induced striatal neurodegeneration comparable to what is seen in C57BL/6 mice. These results suggest that gene(s) on distal chr18 are important determinants of vulnerability to KA-induced hippocampal neurodegeneration, but not QUIN-induced striatal neurodegeneration.

Fibroblast growth factor receptor 3 interacts with and activates TGFß-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFß-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules.

The Huntington's disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a ß-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models.

Animal models of polyglutamine diseases and therapeutic approaches.

The dominant gain-of-function polyglutamine repeat diseases, in which the initiating mutation is known, allow development of models that recapitulate many aspects of human disease. To the extent that pathology is a consequence of disrupted fundamental cellular activities, one can effectively study strategies to ameliorate or protect against these cellular insults. Model organisms allow one to identify pathways that affect disease onset and progression, to test and screen for pharmacological agents that affect pathogenic processes, and to validate potential targets genetically as well as pharmacologically. Here, we describe polyglutamine repeat diseases that have been modeled in a variety of organisms, including worms, flies, mice, and non-human primates, and discuss examples of how they have broadened the therapeutic landscape.

Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes.

Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage.

Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice.

The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntington's disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.