CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice
Introduction
Huntington's disease (HD) is caused by expansions of CAG repeats leading to extended polyglutamine tracts within the corresponding Huntingtin (Htt) protein (Bates et al., 2002, Group, The Huntington's Disease Collaborative Research Group, 1993, Ross, 2002, Walker, 2007). Brain pathology in HD is widespread, particularly in cortex and striatum, however the medium spiny striatal neurons appear to be most vulnerable to mutant Htt expression and show the greatest degree of cell loss (Bates et al., 2002). Neurons and other cell types respond to a wide range of external or internal stimuli by activating cellular signal transduction networks to elicit pro-survival and pro-apoptotic responses (Raman et al., 2007). One such network is the mitogen-activated protein kinase (MAPK) superfamily which is comprised of more than a dozen MAPK genes, the major pathways being the extracellular signal-regulated protein kinases (ERK1/2, ERK), the c-Jun N-terminal kinases or stress-activated protein kinases (JNK/SAPKs), and the p38 family of kinases. Following a cascade of phosphorylation events that initially activate upstream effectors, the individual MAPKs are in turn phosphorylated with subsequent changes in subcellular localization and regulation of downstream targets (Kaneko et al., 1997, McKay and Morrison, 2007, Raman et al., 2007, Turjanski et al., 2007).
MAPK signaling has been implicated in processes contributing to cell death associated with HD and other neurodegenerative diseases. The JNK pathway is activated in several HD systems (Apostol et al., 2006, Garcia et al., 2004, Liu, 1998) and in other neurodegenerative disorders such as Parkinson's and Alzheimer's (Borsello and Forloni, 2007). In mutant Htt-expressing cell systems, genetic or pharmacologic inhibition of JNK signaling blocks Htt-mediated cell death (Apostol et al., 2006, Garcia et al., 2004, Liu, 1998). While the role of JNK in mutant Htt-associated toxicity appears relatively straightforward, that of ERK activation in neurodegenerative diseases is more complex. Both protective (Hetman and Gozdz, 2004) and deleterious (Cheung and Slack, 2004, Chu et al., 2004, Subramaniam and Unsicker, 2006) roles have been ascribed to ERK signaling. These differing functions do not simply rely on ERK's activation state, but also on its relative kinetics, duration of activation, cellular localization and associated scaffold proteins (McKay and Morrison, 2007). ERK activation progressively increases in striatum of R6/2 transgenic mice but decreases in cerebral cortex (Lievens et al., 2002). This latter effect in cortex is consistent with the progressive decrease in brain derived neurotrophic factor (BDNF), a potential downstream target of multiple pathways, including REST/NRSF (Zuccato et al., 2003) and ERK, in HD (Zuccato and Cattaneo, 2007).
A number of proposed mechanisms of HD neurotoxicity involve the progressive depletion of BDNF and the decreased supply of BDNF to striatum through anterograde transport down corticostriatal axons (Gauthier et al., 2004). Remarkably, of all HD mouse models, transcriptional profiles of BDNF knock-out mice are the most similar to gene expression changes identified in HD human brain (Strand et al., 2007), suggesting that decreased BDNF is a key pathogenic feature in HD. Indeed, BDNF treatment of excitotoxic injury mouse models of HD and other means of up-regulating BDNF in HD transgenic mice, including exercise, enriched environments, or treatment with compounds such as cysteamine, have shown promise in ameliorating HD-related symptoms (Borrell-Pages et al., 2006, Levy et al., 2005, Pang et al., 2006, Rigamonti et al., 2007, Saydoff et al., 2006).
Targeted biochemical studies and pharmacologic modulation of MAPK pathways suggest that mutant Htt can alter the metabolic state of cells leading to activation of both ERK and JNK pathways. Activating ERK and JNK could potentially exert competing signals since ERK signaling is associated with cell survival while JNK inhibition effectively suppresses pathogenesis (Apostol et al., 2006). This study tested CEP-11004 and CEP-1347, first characterized as inhibitors of Mixed Lineage Kinases (MLKs) that are upstream activators (MAPKKK) of JNK (Handley et al., 2007, Wang et al., 2004),for their effects on Htt-mediated neuropathogenesis. These inhibitors block JNK activation, but can also activate ERK signaling in certain cell settings (Chadee and Kyriakis, 2004, Roux et al., 2002), and can increase levels of the NGF and BDNF receptors TrkA and TrkB, respectively, in neuronal cells (Wang et al., 2005.). We find that these inhibitors significantly reduced neurotoxicity in cells expressing mutant Htt polypeptides through mechanisms consistent with modulation of JNK and ERK signaling. CEP-1347 also reduces neurotoxicity in striatal cell lines derived from knock-in mice, suppresses neurodegeneration in Drosophila and delays behavioral phenotypes in R6/2 mice. Notably, CEP-1347 increased cortical BDNF expression in R6/2 mice, restoring BDNF levels, suggesting that BDNF expression may be impacted through modulation of MAPK signaling.
Although modulation of MAPK signaling has been implicated previously in HD using proliferating and primary cell culture models (Apostol et al., 2006, Garcia et al., 2004, Roze et al., 2007), modulation of MAPK signaling has not yet been tested in mouse models of HD. These studies take a potential therapeutic compound not previously tested for its effects in any HD model from cells to a pilot preclinical study in mice together with mechanistic insights for its mode of action. We find that selective pharmacologic intervention in MAPK signaling may be an appropriate approach to HD therapy and that CEP-1347, which is safe and well tolerated in humans, represents a promising compound for treatment of HD.
Section snippets
CEP-11004/CEP-1347 protects against mutant Htt-associated phenotypes in PC12 and ST14A cells
We previously reported that the MAP kinases ERK and JNK are activated by expression of mutant Htt in multiple cells lines (Apostol et al., 2006). Activation of JNK paralleled mutant Htt-associated cellular toxicity and a JNK inhibitor (SP600125) partially attenuated increases in caspase 3 activation, suggesting that cell dysfunction/death may involve JNK activation. Conversely, ERK activation was protective and may represent a compensatory mechanism against the deleterious effects of mutant Htt
Discussion
The results presented here suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via the previously unreported mechanism of increasing in vivo BDNF levels using pharmacologic MAPK modulation. CEP-1347 and CEP-11004, MLK inhibitors with neurotrophic properties, inhibit neurotoxicity upon expression of mutant Htt in each model system tested including cells, Drosophila and R6/2 mice. The mechanism of rescue in cell lines is accompanied by modulation of
Propagation of cell lines
Generation and propagation of the Htt14A2.5 line was performed as previously described (Apostol et al., 2003). Htt expression was induced with PA (2.5 or 5 μM) for the indicated times. The ST14A and N548mu and the wild-type STHdhQ7/HdhQ7 and homozygous mutant STHdhQ111/HdhQ111 cell lines were propagated essentially as previously described (Apostol et al., 2006, Cattaneo and Conti, 1998). Briefly, cells were plated in 6-well plates (1 × 105 cells/well) in complete media (DMEM/5% glucose, 10% FBS,
Acknowledgments
This work was supported by the High Q Foundation (to LMT and JLM and DAS), the Hereditary Disease Foundation (to LMT and JLM), Huntington's Disease Society of America Coalition for the Cure (to LMT), NIH awards NS52789 (LMT and JLM), HD36081 (JLM), NS045283 (JLM and LMT), an MBRS NIH award GM55246 (SLW) and Huntington's Disease Society of America Coalition for the Cure and NeuroNe (#512039, FP6, EU) (to EC). We would like to thank Dr. Ira Shoulson for guidance and helpful discussions, Cephalon,
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- 1
Contributed equally to this work.
- 2
Instituto Medicina Molecular, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.