Expanded huntingtin activates the C-JUN N TERMINAL KINASE/C-JUN pathway prior to aggregate formation in striatal neurons in culture

doi:10.1016/j.neuroscience.2004.05.054

Neuroscience

Volume 127, Issue 4, 2004, Pages 859-870

https://doi.org/10.1016/j.neuroscience.2004.05.054 Get rights and content

Abstract

Huntington's disease (HD) is an autosomal neurodegenerative disorder, caused by expansion of a glutamine repeat in the Huntingtin protein. Pathogenesis in HD includes the cytoplasmic cleavage of Huntingtin and release of an amino-terminal fragment capable of nuclear localization, where expanded-Huntingtin (Exp-Htt) might lead to aberrant transcriptional regulation, neuronal dysfunction and degeneration. Recent evidence, from hippocampal cell lines, also implicates altered interaction of Exp-Htt with components of the c-Jun N-terminal kinase (JNK) cascade. However, there is yet no proven implication of the JNK/c-Jun module in degeneration of striatal neurons, the more vulnerable cell population, in HD. In the present study, we used primary striatal neurons in culture to analyze c-Jun activation by Exp-Htt. Green fluorescent protein (GFP)-tagged exon 1 of human Huntingtin either in its normal (25Q, normal-Htt) or expanded (103Q, Exp-Htt) version was transiently transfected in these cells. We first set out, in our conditions, the time course of striatal degeneration produced by Exp-Htt, and found it occurred rapidly. At 48 h post-transfection, 60% of striatal neurons expressing Exp-Htt had apoptotic characteristics including DNA fragmentation and neuritic retraction. Most of these neurons also showed nuclear aggregates of GFP-Exp Htt. Kinetics of c-Jun activation were tested in transfected cells using immunocytochemical detection of phospho-c-Jun. We found a significant activation and induction of c-Jun in Exp-Htt but not normal-Htt-transfected neurons. Of interest, these events occurred prior to nuclear translocation of Exp-Htt. Finally, overexpression of a dominant negative version of c-Jun, as well as pharmacological inhibition of JNK strongly protected against DNA fragmentation and neuritic retraction induced by Exp-Htt. Thus our data suggest that c-Jun activation and induction, is an early event in the pathogenesis of HD, occurring prior to formation of nuclear aggregates of Exp-Htt.

Section snippets

Primary striatal cultures

All experiments on animals were performed in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals. Experiments were designed in order to minimize the number and the suffering of all animals used.

Striata were dissected out from 14 days old embryos from pregnant Swiss mice (Janvier, Le Genest Saint Isle, France). This stage was chosen because it corresponds to the end of the mitotic phase of striatal neurons, and allows the differentiation of these

Results

Primary cultures of striatal neurons were transiently transfected with two constructs encoding the exon 1 of the human Htt containing 25 or 103 glutamine repetition coupled with GFP (GFP-Htt Ex1-25Q, -103Q), and representing the wild type and the mutated forms of the fragment, respectively. These constructs were chosen because i) genetic studies from transgenic mouse model for HD have shown that the expanded exon 1 of human Htt (Exp-Htt Ex1) gene is sufficient to cause a HD-like phenotype (

Discussion

We show in the present work that activation of the JNK/c-Jun module is part of a cascade of events involved in striatal death induced by Exp-Htt exon 1. A significant increase of c-Jun phosphorylation and induction was observed in Exp-Htt exon 1 expressing striatal neurons. In this population, c-Jun activation and induction was an early event in the neurodegenerative process, occurring prior to nuclear aggregate formation. Furthermore, a dominant negative version of c-Jun and a selective JNK

Conclusion

Our data provide the first evidence that inhibition of the JNK/c-Jun module can protect, at least in part, from striatal neuron degeneration in HD. Of course, our data need to be confirmed in more sophisticated model systems, which could take into account the environmental complexity of striatal neurons, for example cortical glutamatergic or nigral dopaminergic afferences. The striatum receives the densest dopaminergic innervation in the brain and dopamine is toxic to striatal neurons in vitro

Acknowledgements

This work was supported by CNRS, University Pierre et Marie Curie and Fondation Schueller-Bethencourt for J. Caboche. M. Garcia was supported by the Ministère de l'Education, de la Recherche et de la Technologie and Fondation pour la Recherche Médicale. D. Charvin was supported by the Ministère de l'Education, de la Recherche et de la Technologie. We acknowledge M. Yaniv for the Δ-c-Jun plasmid, and HDF Resource Bank, UCLA for the GFP tagged versions (25Q and 103Q) of human exon 1 of huntingtin.

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In particular, cultures of striatal neurons from R6/2 HD mice are sensitized to DA-induced oxidative stress, leading to neuronal autophagy (Petersen et al., 2001). ROS produced by low doses of DA potentiate activation of the proapoptotic JNK pathway induced by Exp-HTT (Charvin et al., 2005; Garcia et al., 2004; see Fig. 45.1). Conversely, pharmacological inhibition of this pathway is neuroprotective in the R6/2 transgenic mouse model of HD (Apostol et al., 2008).
Huntington's disease (HD) is the most frequent neurodegenerative disease caused by an expansion of polyglutamines (CAG). The main clinical manifestations of HD are chorea, cognitive, and psychiatric impairments. The transmission of HD is autosomal dominant with a complete penetrance. HD has a single genetic cause, a well-defined neuropathology, and informative premanifest genetic testing of the disease is available. Striatal atrophy begins as early as 15 years before disease onset and continues throughout the period of manifest illness. One important characteristic of HD is the striatal vulnerability, despite similar expression of the protein in other brain areas. Aggregation of the mutated Huntingtin (HTT), excitotoxicity, mitochondrial dysfunctions and energy deficits, alteration of cholesterol metabolism along with transcriptional dysregulation are all part of the cellular events that underlie neuronal dysfunction. Among these nonexclusive mechanisms, an alteration of cortico-striatal signaling is thought to orchestrate the downstream events involved in the cascade of striatal dysfunction.
Ethyl pyruvate ameliorates 3-nitropropionic acid-induced striatal toxicity through anti-neuronal cell death and anti-inflammatory mechanisms
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The potential neuroprotective value of ethyl pyruvate (EP) for the treatment of the striatal toxicity is largely unknown. We investigated whether EP promotes the survival of striatal neurons in a 3-nitropropionic acid (3-NP)-induced mouse model of Huntington’s disease (HD). EP (5, 10, 20, and 40 mg/kg/day, i.p.) was daily injected from 30 min before 3-NP intoxication (pretreatment) and from onset/progression/peak point of neurological impairment by 3-NP intoxication. EP produced a neuroprotective effect in dose- and time-dependant manners. EP pretreatment of 40 mg/kg/day produced the best neuroprotective effect among other conditions. Pretreatment of EP significantly attenuated neurological impairment and lethality and prevented formation of lesion area and neuronal loss in the striatum after 3-NP intoxication. This neuroprotection afforded by EP was associated with the suppression of succinate dehydrogenase activity, apoptosis, and microglial activation. The suppressive effect of EP corresponded to the down-regulation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signal pathways, and mRNA expression of inflammatory mediators including tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the striatum after 3-NP intoxication. Interestingly, the intrathecal introduction of inhibitors MAPKs and NF-κB into control mice decreased the lethality after 3-NP intoxication. Our findings indicate that EP may effectively alleviate 3-NP-induced striatal toxicity by inhibition of the MAPKs and NF-κB pathways in the striatum, and that EP has a wide therapeutic window, suggesting that EP may have therapeutic value in the treatment of aspects of HD’s disease related to inflammation.
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Citation Excerpt :
It is caused by the expansion of an unstable CAG trinucleotide repeat within the first exon of the IT-15 gene encoding huntingtin (Htt) protein [208,209]. The function of Htt protein has not been completely elucidated; however, it is possibly involved in endocytosis, vesicular trafficking [210], embryonic development [211] and transcriptional regulation [212]. The CAG repeat in Htt shows between 10 and 29 copies in healthy subjects and it is expanded to 36–121 in HD [213].
Neurodegenerative diseases constitute a worldwide health problem. Metals like iron and copper are essential for life, but they are also involved in several neurodegenerative mechanisms such as protein aggregation, free radical generation and oxidative stress. The role of Fe and Cu, their pathogenic mechanisms and possible therapeutic relevance are discussed regarding four of the most common neurodegenerative diseases, Alzheimer's, Parkinson's and Huntington's diseases as well as amyotrophic lateral sclerosis. Metal-mediated oxidation by Fenton chemistry is a common feature for all those disorders and takes part of a self-amplifying damaging mechanism, leading to neurodegeneration. The interaction between metals and proteins in the nervous system seems to be a crucial factor for the development or absence of neurodegeneration. The present review also deals with the therapeutic strategies tested, mainly using metal chelating drugs. Metal accumulation within the nervous system observed in those diseases could be the result of compensatory mechanisms to improve metal availability for physiological processes.
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Huntington's disease (HD) is a neurodegenerative disorder resulting from the expansion of a glutamine repeat (polyQ) in the N-terminus of the huntingtin (htt) protein. Expression of polyQ-containing proteins has been previously shown to induce various cellular stress responses. Among these, activation of the c-Jun N-terminal kinase (JNK) cascade has been observed in cellular models of HD. However, the implication of the JNK pathway has not previously been evaluated in the striatum of HD animal models. Here we report that the JNK pathway participates in HD pathology in a rat model of the disease. Increased phosphorylation of the JNK target c-Jun was observed as early as 4 weeks and persisted for 13 weeks after lentiviral-mediated expression of htt171-82Q. In order to assess the importance of this pathway in HD pathology, JNK inhibitors including dominant-negative mutants of upstream kinases (ASK1^K709R, MEKK1^D1369A), a c-Jun mutant (Δ169c-Jun) and the active domain of the scaffold protein JIP-1/IBI (IBI-JBD) were tested for their ability to mitigate the effect of htt171-82Q. The overexpression of MEKK1^D1369A and JIP-1/IBI reduced the polyQ-related loss of DARPP-32 expression, while the other inhibitors had no effect. In all cases, the formation of EM48-positive htt inclusions and P-c-Jun immunoreactivity were unaltered. These results suggest that JNK activation is involved in HD and that blockade of this pathway may be of benefit in counteracting HD-related neurotoxicity.
CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice
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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.
Granulocyte-colony stimulating factor attenuates striatal degeneration with activating survival pathways in 3-nitropropionic acid model of Huntington's disease
2008, Brain Research
Citation Excerpt :
3NP induces JNK activation and c-Jun expression in the striatum and 3NP-induced striatal death is controlled by the activation of the JNK pathway (Garcia et al., 2002). In HD, mutated huntingtin can activate JNK in hippocampal cells (Liu et al., 2000; Apostol et al., 2007) and in striatal neuronal cells via mechanisms similar to those observed in 3NP experimental models (Garcia et al., 2002; Garcia et al., 2004). Damage to mitochondria by neurotoxins forms more ROS from the electron transporter chain and provokes oxidative damage that modifies proteins and other biomolecules (Halliwell, 2006).
Huntington's disease (HD) has a mitochondrial dysfunction causing the vulnerability to the excitotoxicity and activations of multiple cell death pathways. Recent evidences suggest that the hematopoietic cytokine, granulocyte-colony stimulating factor (G-CSF), exerts pleiotropic neuroprotection in acute neural injury with activating various survival pathways. Thus, we investigated whether G-CSF can modulate neurodegeneration in an HD animal model induced by 3-nitropropionic acid (3NP), which inhibits mitochondrial succinate dehydrogenase complex II. Either G-CSF (50 μg/kg/day) or saline (as vehicle) was administered intraperitoneally for 5 days with 3NP (63 mg/kg/day) continuous osmotic pump infusion into male Lewis rats. We measured motor scales (0–8) daily and sacrificed rats at 5 days. We observed that G-CSF receptors were expressed in 3NP-induced degenerating striatum. Rats treated with G-CSF showed less degree of neurologic deficits. In the G-CSF-treated rats, the striatal lesion volume measured by Nissl staining, TUNEL⁺ apoptotic cells, Fluorojade C⁺ degenerating neurons, and c-Jun⁺ cells were all decreased. In western blotting, G-CSF activated survival pathways including p-ERK, p-eNOS, p-STAT3, and p-Akt. In summary, G-CSF was found to have neuroprotective effects and save striatal cells through activations of survival pathways in the 3NP-induced striatal degeneration model for HD.

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¹: Present address: Venetian Institute of Molecular Medicine and Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy.

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Expanded huntingtin activates the C-JUN N TERMINAL KINASE/C-JUN pathway prior to aggregate formation in striatal neurons in culture

Abstract

Section snippets

Primary striatal cultures

Results

Discussion

Conclusion

Acknowledgements

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