Elsevier

Neuropharmacology

Volume 56, Issue 2, February 2009, Pages 422-428
Neuropharmacology

Evidence of calpain/cdk5 pathway inhibition by lithium in 3-nitropropionic acid toxicity in vivo and in vitro

https://doi.org/10.1016/j.neuropharm.2008.09.012Get rights and content

Abstract

Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.

Introduction

Lithium salts are the most commonly used drug to treat affective disorders, mainly bipolar disorder (Manji et al., 1999a, Manji et al., 1999b, Shaldubina et al., 2001). Moreover, a new potential application of lithium as a neuroprotective agent has emerged (Li et al., 2002). This drug protects against apoptosis induced by a variety of insults in cultured cells, as well as in animal models of various neurodegenerative diseases. These insults include glutamate (Shao et al., 2005), β-amyloid (Alvarez et al., 2002), colchicine (Jordà et al., 2004) and ceramide (Centeno et al., 1998) treatment, withdrawal of growth factor (Bhat et al., 2000, Yeste-Velasco et al., 2007), and deprivation of potassium (Mora et al., 2001, Mora et al., 2002). In vivo studies also indicate that lithium has neuroprotective properties. It reduces irradiation-induced cerebellar degeneration (Inouye et al., 1995), quinolinic acid-induced lesions in rat striatum (Wei et al., 2001), focal ischemia-induced infarction in rat brains (Nonaka and Chuang, 1998) and prevents neurotoxicity in in vivo models of Parkinson's diseases (Youdim and Arraf, 2004).

The mechanisms underlying lithium neuroprotection involve activation of the phosphatidylinositol 3-kinase/Akt signaling pathway (Chalecka-Franaszek and Chuang, 1999), decreased expression of pro-apoptotic proteins such as p53 and Bax (Chen and Chuang, 1999) and enhanced expression of the anti-apoptotic protein Bcl-2 (Chen et al., 1999, Chen and Chuang, 1999). Moreover, lithium modulates NMDA receptor activity and reduces the intracellular calcium increase mediated by NMDA receptor stimulation (Hashimoto et al., 2002). Despite all these data, the exact mechanisms and molecular links between these multiple neuroprotective mechanisms remain unclear.

In an attempt to elucidate the molecular mechanisms underlying the neuroprotective effects of lithium, we focused our study on the 3-nitropropionic acid (3-NP) model. 3-NP is an inhibitor of succinate dehydrogenase (SDH), a Complex II respiratory enzyme required for mitochondrial energy production, and its administration in vivo induces a selective striatal pathology similar to that observed in Huntington's disease (HD) providing a useful experimental model of this pathology (Beal et al., 1993, Brouillet et al., 1999). Mitochondrial dysfunction is heavily involved in the ageing process and a considerable body of evidence indicates that this process makes a crucial contribution to the pathophysiologic mechanism of neurodegeneration (Calabrese et al., 2001, Beal, 2007, Oliveira et al., 2007).

Several studies have addressed the participation of calpain in 3-NP toxicity. It has been proposed that calpain is the essential cell death effector in 3-NP-induced HD-like pathogenesis and it is now clear that calpain activation is an early process in 3-NP-induced neuronal cell death (Bizat et al., 2003). During the last decade, extensive research has revealed the deregulation of calpain activity as a key cytotoxic event in a variety of neurodegenerative disorders such as postischemic neurodegeneration, Alzheimer's, Parkinson's and HD (Crocker et al., 2003, Gafni and Ellerby, 2002, Saito et al., 1993, Yokota et al., 1995). Furthermore, the abnormal activation of calpain attenuated by overexpresion of calpastatin, the natural calpain inhibitor, or by other calpain inhibitors provides marked neuroprotection in the 3-NP model (Bizat et al., 2003, Lee et al., 1991, Arlinghaus et al., 1991, Higuchi et al., 2005). Cdk5-activating protein p35 is cleaved by calpain into a p25 isoform that causes unregulated cdk5 activity and neurodegeneration (Patrick et al., 1999, Lee et al., 2000, Camins et al., 2006).

Interestingly, cdk5/p25 phosphorylates the nuclear transcription myocite enhancing factor 2 (MEF2), thereby inhibiting its pro-survival activity (Camins et al., 2006). Recently, we described a relationship between neuronal cell loss induced by 3-NP and strong activation of cdk5 by calpain, which, in turn, reduces the neuroprotective effect mediated by MEF2 (Crespo-Biel et al., 2007). Cdk5 is the only cyclin-dependent kinase not related to cell cycle regulation. A recent report described 3-NP-induced cell cycle activation in striatal neurons after calpain activation, and was the first study to demonstrate that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain (Akashiba et al., 2008).

Since increased levels of active forms of calpain have been found in tissue from patients with neurodegenerative diseases, it has been hypothesized that blockade of this cysteine protease may be of therapeutic interest (Di Rosa et al., 2002). Thus, it is of relevance to identify downstream targets gated to calpain in order to find an adequate or potential treatment for neurodegenerative diseases as HD.

Here we examined the neuroprotection effect of lithium treatment on a model of HD, the 3-NP model, to determine whether treatment with this drug inhibits calpain and cdk5 activation induced by this mitochondrial toxin and thus whether lithium provides a protective effect against the striatal lesion present. We demonstrate a novel mechanism of lithium in inhibiting cell death by preventing calpain/cdk5 activation.

Section snippets

Animal treatment

The animal experiments met the guidelines endorsed by the Ethical Committee of the University of Barcelona, which is under the supervision of the Generalitat de Catalunya (Autonomous Government of Catalonia). All efforts were made to reduce the number of animals used and to minimize suffering. For in vivo studies, we used two-month-old male Sprague–Dawley rats, each weighing 180–220 g. Animals were group-housed under a 12 h light/dark cycle in a vivarium and provided with food and water ad

Effects of lithium on 3-NP-induced mortality and general health of the animals

Lithium concentration in blood was 0.9 mEq/l, thereby indicating that treatment reached the minimal therapeutic concentration to exert beneficial effects.

The effects of lithium on 3-NP-induced mortality were also examined. The survival rate was significantly higher in the 3-NP + Li group than the 3-NP group. Only 30% of the animals treated with 3-NP alone survived, whereas 68% of the 3-NP + Li group survived after five consecutive days of treatment. Thus, lithium treatment significantly

Discussion

No proven medical treatment is currently available to mitigate the devasting clinical manifestation of HD. During recent years intensive research efforts have been dedicated to this field. Lithium has been widely used as a treatment of bipolar disorders since 1950. However, despite its efficacy, the therapeutic targets of this drug remain unknown. Therefore, here we examined the efficacy of lithium to prevent neurodegeneration of the striatum in 3-NP rat model and the possible participation of

Acknowledgments

This work was supported by grants SAF-2006-13092 from the Ministerio de Educación y Ciencia (Spain); PI041300 and Centros de Investigación Biomédica en Red (CIBER) from the Instituto de Salud Carlos III; 2005/SGR00893 from the Generalitat de Catalunya; la Obra Social Caixa de Sabadell and la Fundació La Marató 063203.

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