BioMed Central Page 1 of 11 (page number not for citation purposes) Molecular Brain Open Access Research Genetic and pharmacological inhibition of calcineurin corrects the BDNF transport defect in Huntington's disease Jose R Pineda1,2, Ra��l Pardo1,2, Diana Zala1,2, Hua Yu1,2, Sandrine Humbert1,2 and Fr��d��ric Saudou*1,2 Address: 1Institut Curie, Unit�� Mixte de Recherche 146, F-91405 Orsay, France and 2Centre National de la Recherche Scientifique, Unit�� Mixte de Recherche 146, F-91405 Orsay, France Email: Jose R Pineda - Jose-Ramon.Pineda-Marti@curie.u-psud.fr Ra��l Pardo - Raul.Pardo@curie.u-psud.fr Diana Zala - Diana.Zala@curie. u-psud.fr Hua Yu - Hua.Yu@curie.u-psud.fr Sandrine Humbert - Sandrine.Humbert@curie.fr Fr��d��ric Saudou* - Frederic.saudou@curie. u-psud.fr * Corresponding author Abstract Background: Huntington's disease (HD) is an inherited neurogenerative disease caused by an abnormal expansion of glutamine repeats in the huntingtin protein. There is currently no treatment to prevent the neurodegeneration caused by this devastating disorder. Huntingtin has been shown to be a positive regulator of vesicular transport, particularly for neurotrophins such as brain- derived neurotrophic factor (BDNF). This function is lost in patients with HD, resulting in a decrease in neurotrophic support and subsequent neuronal death. One promising line of treatment is therefore the restoration of huntingtin function in BDNF transport. Results: The phosphorylation of huntingtin at serine 421 (S421) restores its function in axonal transport. We therefore investigated whether inhibition of calcineurin, the bona fide huntingtin S421 phosphatase, restored the transport defects observed in HD. We found that pharmacological inhibition of calcineurin by FK506 led to sustained phosphorylation of mutant huntingtin at S421. FK506 restored BDNF transport in two complementary models: rat primary neuronal cultures expressing mutant huntingtin and mouse cortical neurons from HdhQ111/Q111 HD knock-in mice. This effect was the result of specific calcineurin inhibition, as calcineurin silencing restored both anterograde and retrograde transport in neurons from HdhQ111/Q111 mice. We also observed a specific increase in calcineurin activity in the brain of HdhQ111/Q111 mice potentially accounting for the selective loss of huntingtin phosphorylation and contributing to neuronal cell death in HD. Conclusion: Our results validate calcineurin as a target for the treatment of HD and provide the first demonstration of the restoration of huntingtin function by an FDA-approved compound. Background An abnormal polyglutamine (polyQ) expansion in the N- terminal part of the huntingtin protein causes Hunting- ton's disease (HD), a fatal neurodegenerative disorder characterized by the dysfunction and death of striatal and cortical neurons in the brain [1]. HD is characterized by motor, cognitive and psychiatric symptoms and the age at onset is inversely correlated with the number of CAGs encoding glutamines in the huntingtin protein. There is currently no effective treatment for preventing the death Published: 27 October 2009 Molecular Brain 2009, 2:33 doi:10.1186/1756-6606-2-33 Received: 18 September 2009 Accepted: 27 October 2009 This article is available from: http://www.molecularbrain.com/content/2/1/33 �� 2009 Pineda et al licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Brain 2009, 2:33 http://www.molecularbrain.com/content/2/1/33 Page 2 of 11 (page number not for citation purposes) of neurons in the brain or disease progression. Promising treatment strategies involve the identification of com- pounds capable of restoring functions altered in disease [2]. The mechanisms underlying neuronal dysfunction and death in HD are complex and involve both a gain of new toxic functions and a loss of the neuroprotective functions of wild-type huntingtin [1]. Several groups have demon- strated changes in the microtubule (MT)-dependent trans- port of vesicles, such as those containing brain-derived neurotrophic factor (BDNF), in diseased neurons [3-7]. This trafficking defect is an early pathogenic event and is linked to the association of huntingtin with components of the molecular motor machinery [3,8-13] and its func- tion as a direct regulator of MT-dependent transport in different cell type including neurons [3,10,12]. Huntingtin phosphorylation at S421 abolishes the toxic- ity of mutant huntingtin in vitro and in vivo [14,15]. We recently demonstrated that the phosphorylation of mutant huntingtin at the S421 residue promotes neuro- protection in HD, by restoring huntingtin function in the transport of BDNF [16]. In particular, we found that path- ogenic polyQ-huntingtin with an S421 mutation mimick- ing constitutive phosphorylation transports vesicles as efficiently as the wild-type protein. However, the potential benefits of drugs promoting huntingtin S421 phosphor- ylation and abolishing the transport defect in HD remain to be evaluated. Huntingtin phosphorylation at S421 is induced by the IGF-1/Akt pathway and inhibited by cal- cineurin [14,15]. Lower than normal levels of huntingtin phosphorylation are found in various HD models [15,17]. These lower levels of phosphorylation may be due to changes in Akt during disease progression, as observed in animal models and in the brains of HD patients [14,18] and/or an increase in calcineurin activity [15]. Consistent with this hypothesis, calcineurin levels have been found to be higher than normal in neuronal cells immortalized from HD mice [19]. A decrease in the levels of RCAN1-1L, a negative regulator of calcineurin, in the brains of HD patients may also account for the lower levels of huntingtin phosphorylation observed [20]. These observations suggest that calcineurin inhibition may be of benefit in the treatment of HD. Calcineurin is a serine-threonine phosphatase that is highly abundant in neuronal tissues. It consists of a cal- modulin-binding 60 kDa catalytic subunit, calcineurin A (CaNA), and an intrinsic Ca+2-binding 19 kDa regulatory subunit, calcineurin B (CaNB) [21-24]. The C-terminal part of CaNA contains autoinhibitory and calmodulin- binding domains and this subunit is regulated by various endogenous regulators, including RCAN proteins [25-30]. Calcineurin is also efficiently blocked by FK506, an immunosuppressive drug that must bind to FK506-bind- ing proteins to exert its effects. FK506 has been shown to be neuroprotective in various neurodegenerative para- digms [31,32]. In this study, we investigated the potential value of cal- cineurin as a target for the treatment of HD by pharmaco- logical and silencing approaches. Calcineurin activity was found to be dysregulated in the brains of HD mice. FK506 and siRNAs targeting calcineurin increased huntingtin phosphorylation, restoring the capacity of this protein to transport BDNF in neurons to levels similar to those in the wild-type. Thus, drugs or pathways blocking calcineurin activity are of potential interest for the treatment of HD. Results FK506 increases huntingtin phosphorylation at S421 in primary cortical neurons from HdhQ111/Q111 mice Previous studies have demonstrated that calcineurin dephosphorylates the S421 residue of huntingtin in rat cultures in vitro and that calcineurin inhibition results in an increase in huntingtin phosphorylation in transfected cells [15]. As a first step towards validating calcineurin as a treatment target, we investigated whether endogenous mutant huntingtin could be phosphorylated in neurons. We established primary cultures of neurons from HdhQ111/ Q111 embryos and analyzed the extent to which various concentrations of FK506 induced the selective phosphor- ylation of huntingtin at S421, using a highly specific S421-phospho-htt antibody [15]. The treatment of pri- mary neurons for one hour with FK506 at a concentration of at least 0.3 ��M induced a significant increase in the selective phosphorylation of endogenous mutant hunt- ingtin, as shown by Western blotting (Figure 1A). We expressed huntingtin phosphorylation as the ratio of phosphorylated to total huntingtin, using ��-tubulin as a loading control, and found that increasing FK506 concen- tration induced progressive, strong phosphorylation at S421. A significant increase in phosphorylation was observed from concentrations as low as 0.1 ��M (Figure 1B). FK506 corrects the polyQ-huntingtin-induced defect in BDNF transport in rat cortical neurons We previously demonstrated that a mutation mimicking constitutive phosphorylation at S421 (a serine to aspartic acid substitution) restored the capacity of transfected mutant polyQ-huntingtin to transport BDNF at velocities similar to those achieved by the wild-type protein [13]. We investigated whether FK506 could rescue the transport defect induced by the polyQ expansion, by analyzing the dynamics of BDNF-mCherry-containing vesicles in rat pri- mary cultures by fast 3D videomicroscopy followed by deconvolution, as previously described [3,12,13,33]. Video experiments were performed three days after the electroporation of primary cultures of embryonic E17 rat cortical neurons. We checked that the differences in trans-