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.

SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.

A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract within the huntingtin protein (Htt). Identifying the pathways that are altered in response to the mutant protein is crucial for understanding the cellular processes impacted by the disease as well as for the rational development of effective pharmacological interventions. Here, expression profiling of a cellular HD model identifies genes that implicate altered mitogen-activated protein kinase (MAPK) signaling. Targeted biochemical studies and pharmacological modulation of these MAPK pathways suggest that mutant Htt affects signaling at upstream points such that both ERK and JNK are activated. Modulation of the ERK pathway suggests that this pathway is associated with cell survival, whereas inhibition of JNK was found to effectively suppress pathogenesis. These studies suggest that pharmacological intervention in MAPK pathways, particularly at the level of ERK activation, may be an appropriate approach to HD therapy.

Sustained ERK1/2 but not STAT1 or 3 activation is required for thanatophoric dysplasia phenotypes in PC12 cells.

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of short-limbed dwarfism, achondroplasia (ACH), as well as neonatal lethal forms, thanatophoric dysplasia (TD) I and II. The causative mutations induce graded levels of constitutive activation of the receptor that correspond to the severity of the disorder, resulting in premature entry into hypertrophic differentiation and reduced proliferation of chondrocytes in developing cartilage. Although FGFR3 promotes growth in most tissues, it is a negative regulator of endochondral bone growth. Several signaling pathways have been implicated in these skeletal disorders including the Ras/MEK/ERK pathway and the JAK/STAT, the latter in the most severe phenotypes, however their functional relevance remains incompletely understood. Using PC12 cell lines stably expressing inducible mutant receptors containing the TDII mutation, K650E, sustained activation of ERK1/2 and activation of STAT1 and STAT3, but not STAT5, is observed in the absence of ligand. This activation leads to neurite outgrowth, a phenotypic readout of constitutive receptor activity, and sustained ERK1/2 activity is required for this ligand-independent differentiation. To assess the functional relevance of STAT activation induced by the mutant receptor, STATs were specifically downregulated using RNA-interference. Silencing of STAT1 or 3 independently or in combination had no significant effect on ligand-independent neurite outgrowth, ERK1/2 activation or p21(WAF1/CIP1) protein levels. These results support a model in which sustained activation of ERK1/2 is a key regulator of the increased transition to hypertrophic differentiation of the growth plate, whereas activation of STATs 1 and 3 is not required.

Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila.

We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the Drosophila CNS. We use a Drosophila Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in Drosophila to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.

Autophagy regulates the processing of amino terminal huntingtin fragments.

The N-terminus of mutant huntingtin (htt) has a polyglutamine expansion and forms neuronal aggregates in the brain of Huntington's disease (HD) patients. Htt expression in vitro activates autophagy, but it is unclear whether autophagic/lysosomal pathways process htt, especially N-terminal htt fragments. We explored the role of autophagy in htt processing in three cell lines, clonal striatal cells, PC12 cells and rodent embryonic cells lacking cathepsin D. Blocking autophagy raised levels of exogenously expressed htt1-287 or 1-969, reduced cell viability and increased the number of cells bearing mutant htt aggregates. Stimulating autophagy promoted htt degradation, including breakdown of caspase cleaved N-terminal htt fragments. Htt expression increased levels of the lysosomal enzyme cathepsin D by an autophagy-dependent pathway. Cells without cathepsin D accumulated more N-terminal htt fragments and cells with cathepsin D were more efficient in degrading wt htt than mutant htt in vitro. These results suggest that autophagy plays a critical role in the degradation of N-terminal htt. Altered processing of mutant htt by autophagy and cathepsin D may contribute to HD pathogenesis.

Complex alteration of NMDA receptors in transgenic Huntington's disease mouse brain: analysis of mRNA and protein expression, plasma membrane association, interacting proteins, and phosphorylation.

We analyzed NMDA receptor subunit mRNAs, proteins, and anchoring proteins in mice transgenic for exon 1 of the HD gene. R6/2 mice had decreased levels of mRNAs encoding epsilon1 and epsilon2 NMDA receptor subunits (mouse orthologs of rat NR2A and NR2B subunits), but not the zeta1 subunit (mouse ortholog of NR1), as assessed by gene expression profiling and Northern blotting. In situ hybridization resolved mRNA decreases spatially to the CA1 field of hippocampus. Western blotting revealed decreases in plasma membrane-associated epsilon1 and epsilon2 subunits in hippocampus, and decreases in plasma membrane-associated zeta1 subunit in cortex and hippocampus. In addition, PSD-95 and alpha-actinin-2, proteins essential for anchoring NMDA receptors, were decreased. Finally, we found a decreased level of tyrosine-phosphorylated epsilon1 subunit, another determinant of NMDA receptor trafficking, in R6/2 hippocampus. Taken together, these data demonstrate multiple levels of NMDA receptor dysregulation, including abnormalities in mRNA expression levels, receptor stoichiometry, protein phosphorylation, and receptor trafficking.

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.