Genetic and pharmacological inhibition of calcineurin corrects the BDNF transport defect in Huntington's disease
© Pineda et al; licensee BioMed Central Ltd. 2009
Received: 18 September 2009
Accepted: 27 October 2009
Published: 27 October 2009
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.
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.
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.
An abnormal polyglutamine (polyQ) expansion in the N-terminal part of the huntingtin protein causes Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the dysfunction and death of striatal and cortical neurons in the brain . 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 of neurons in the brain or disease progression. Promising treatment strategies involve the identification of compounds capable of restoring functions altered in disease .
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 . Several groups have demonstrated changes in the microtubule (MT)-dependent transport 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 function as a direct regulator of MT-dependent transport in different cell type including neurons [3, 10, 12].
Huntingtin phosphorylation at S421 abolishes the toxicity of mutant huntingtin in vitro and in vivo [14, 15]. We recently demonstrated that the phosphorylation of mutant huntingtin at the S421 residue promotes neuroprotection in HD, by restoring huntingtin function in the transport of BDNF . In particular, we found that pathogenic polyQ-huntingtin with an S421 mutation mimicking constitutive phosphorylation transports vesicles as efficiently as the wild-type protein. However, the potential benefits of drugs promoting huntingtin S421 phosphorylation 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 calcineurin [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 . Consistent with this hypothesis, calcineurin levels have been found to be higher than normal in neuronal cells immortalized from HD mice . 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 . 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 calmodulin-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-binding proteins to exert its effects. FK506 has been shown to be neuroprotective in various neurodegenerative paradigms [31, 32].
In this study, we investigated the potential value of calcineurin as a target for the treatment of HD by pharmacological 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.
FK506 increases huntingtin phosphorylation at S421 in primary cortical neurons from HdhQ111/Q111 mice
FK506 corrects the polyQ-huntingtin-induced defect in BDNF transport in rat cortical neurons
We then investigated the effects of the various constructs on BDNF vesicular transport. As previously reported [3, 12, 13, 33], an N-terminal 480-amino acid fragment of huntingtin with a wild-type glutamine stretch (480-17Q) stimulated transport, whereas this function was lost if the huntingtin contained the pathological expanded polyQ stretch (Figure 2C-F). In particular, we observed significant effects of the polyQ expansion on both antero- and retrograde movement and on other dynamic parameters, such as the percentage of pausing time (Figure 2E) and the total distance traveled (Figure 2F). Our findings confirm that the wild-type huntingtin fragment reproduces the transport function of the full-length protein . We next investigated whether FK506 could correct the mutant huntingtin-induced transport defect. Neurons were maintained in Neurobasal B27 serum-free medium to prevent high basal levels of huntingtin phosphorylation at S421. One hour of treatment with FK506 at a concentration inducing maximal levels of huntingtin phosphorylation (Figure 1A) restored dynamic parameters to control levels (Figure 2C-F and Supplemental Movie 1). In particular, FK506 significantly increased BDNF transport velocities in 480-68Q expressing neurons to 480-17Q levels, for both anterograde values (480-17Q + DMSO: 0.41 ± 0.02 μm/s; 480-68Q + DMSO: 0.26 ± 0.02 μm/s; 480-68Q + FK506 1 μM: 0.45 ± 0.03 μm/s; Tukey HSD p < 0.0002)(Figure 2C) and retrograde values (480-17Q + DMSO: 0.45 ± 0.07 μm/s; 480-68Q + DMSO: 0.27 ± 0.02 μm/s; 480-68Q + FK506 1 μM: 0.45 ± 0.07 μm/s; Tukey HSD p < 0.0002) (Figure 2D). FK506 treatment also significantly decreased the pausing time of 480-68Q-electroporated neurons (91.55 ± 6.87%) to a value (47.46 ± 4.84%) within the range for 480-17Q electroporated neurons (44.18 ± 3.80%, Tukey HSD p < 0.0002) (Figure 2E). Strikingly, we also observed a significant effect of FK506 on the total distance covered by vesicles (Figure 2F).
The addition of 1 μM FK506 to cells expressing wild-type huntingtin did not significantly increase anterograde or retrograde velocities, consistent with huntingtin being highly phosphorylated in wild-type conditions, with no further phosphorylation possible upon calcineurin inhibition (Figure 2C-F). Thus, FK506 corrects the BDNF transport defect in neurons expressing mutant huntingtin.
FK506 restores BDNF transport in cortical neurons from HdhQ111/Q111 mice
FK506 inhibits calcineurin activity in cortical neurons from HdhQ111/Q111 mice
Genetic inhibition of calcineurin restores BDNF transport in cortical neurons from HdhQ111/Q111 mice
HD mice display high levels of calcineurin activity
We demonstrate here that calcineurin is dysregulated in Huntington's disease and that the pharmacological and genetic inactivation of calcineurin leads to an increase in the phosphorylation of mutant huntingtin at S421, resulting in the restoration of its function in the intracellular transport of BDNF within cortical neurons.
Our results provide the first demonstration of a direct role of calcineurin in the regulation of vesicular transport. Huntingtin regulates the MT-dependent transport of organelles in neurons [3, 10, 12, 33] and this function is regulated by phosphorylation in both physiological and pathological conditions [12, 13]. We found that mutations mimicking huntingtin phosphorylation restored the ability of mutated huntingtin to transport BDNF. Also, we found that promoting huntingtin phosphorylation by activating the IGF-1/Akt pathway, leading to huntingtin phosphorylation at S421, restored the function of the mutant huntingtin protein in MT-dependent transport . Our results demonstrating that calcineurin inhibition increases S421 phosphorylation and transport also provide support for the notion that huntingtin phosphorylation at S421 is critical for regulation of the function of this protein in transport and for neuronal death in HD [12, 13]. Consistent with these data, FK506 exerts neuroprotective effects in neurons expressing polyQ-huntingtin . Our observation that calcineurin inhibition restores BDNF trafficking and supply in the striatum in HD is consistent with previous studies showing that calcineurin inhibitors, such as cyclosporin A and FK506, decrease the level of neuronal cell death in the hippocampus after forebrain ischemia in animal models [41, 42]. BDNF was reported to mediate the neuroprotective effect of calcineurin inhibitors, as shown by the selective induction of BDNF in the hippocampus of cyclosporine treated animals . The induction of BDNF was linked to the increase in pCREB levels and BDNF transcription, but our results suggest that calcineurin inhibition could also increase BDNF trophic support by stimulating the transport of vesicles containing BDNF. Our results indicate that further studies of the general role of calcineurin in the control of intracellular trafficking in health and disease are warranted.
Our results validate calcineurin as a target for treatment in HD. We have demonstrated a specific change in calcineurin activity in disease conditions. Indeed, although calcineurin protein levels are similar in mouse brains containing the wild-type and mutant HD, significantly higher levels of calcineurin activity were observed in the cortex of mutant mice. This dysregulation is consistent with the downregulation of RCAN1-1L, a negative regulator of calcineurin in brains affected by HD  and the disturbance of calcium concentrations in HD . In addition, activation of calcineurin could be induced by the cleavage of its specific inhibitor cain/cabin1 following the activation of the protease calpain . Indeed, calpain activity is increased in HD brains and contributes to HD pathology [46, 47]. Further evidence for the specific dysregulation of calcineurin in HD is provided by our observation that the treatment of neurons expressing wild-type huntingtin with FK506 had no effect on vesicular transport, whereas this treatment had a strong effect in neurons expressing the mutant protein. We have also shown that calcineurin activity is strongly inhibited by FK506 in neurons from HD mice. Thus, drugs aiming to block calcineurin activity are likely to be effective at treating the disease. Finally, the inhibition of calcineurin through two approaches, pharmacological and genetic in nature, resulted in similar beneficial effects, with an increase in the levels of phosphorylated huntingtin and correction of the axonal transport defect observed in HD.
This study sheds light on the molecular mechanism by which calcineurin inhibition blocks neuronal death in HD. Our results extend the therapeutic potential of calcineurin inhibitors such as FK506, an FDA-approved drug capable of crossing the blood-brain barrier, and provide evidence in favour of clinical trials of the use of such compounds in HD patients.
Statview 4.5 software (SAS Institute Inc., Cary, NC) was used for statistical analysis. Data are expressed as mean +/- S.E.M.
Constructs and siRNA
The plasmid encoding BDNF-mCherry was a generous gift from G. Banker (Oregon Health and Science University, Portland, Oregon). BDNF-mCherry shows cellular localization, processing, and secretion properties indistinguishable from those of endogenous BDNF. The wild-type and polyQ huntingtin constructs 480-17Q, 480-68Q, have been previously described  and correspond to human-mouse hybrids derived from mouse huntingtin cDNA: first exon of mouse huntingtin has been substituted by the homologous human one in mouse full length cDNA . The siRNAs targeting mouse huntingtin correspond to the coding region 361-380 (siRNA1) of huntingtin mouse mRNA (GenBank Acc. n° XM_132009). The siRNA sequences targeting rat CaNAα and CaNAβ correspond to the coding regions 677-695 (GenBank Acc. No. NM 017041) and 448-466 (GenBank Acc. No. NM 017042) respectively. The scramble RNA (scRNA) control (Eurogentec, Seraing, Belgium) used has a unique sequence which does not match to any sequence in the genome of interest.
Except for the experiments assessing BDNF transport in rat cortical neurons, all the videomicroscopy experiments and the biochemical analyses were conducted using HdhQ111/+ HdhQ111/Q111 and the corresponding Hdh+/+ mice in the CD1 background. HdhQ111 knock-in mice, a generous gift from M.E. MacDonald, have been previously described . For neuronal cultures from rat embryos, time pregnant Sprague-Dawley rats were obtained from Charles River Laboratories (Les Oncins, France). For experiments requiring brain dissection (Western blotting analyses and calcineurin assays), mice were deeply anaesthetized in a CO2 chamber, and their cortices, substantia nigra and striata were dissected out on ice and rapidly frozen using CO2 pellets. All experimental procedures were performed in strict accordance with the recommendations of the European Community (86/609/EEC) and the French National Committee (87/848) for care and use of laboratory animals.
Neuronal cultures and transfection
Primary cortical neurons from E17 rat or from HdhQ111/Q111 or Hdh+/+ mouse E15 embryos were prepared, cultured in Neurobasal B27 and transfected as described [3, 14, 48]. Cortical neurons were electroporated with the rat neuron Nucleofector® kit according to the supplier's manual (Amaxa, Biosystem, Köln, Germany). For huntingtin gene replacement strategy, neurons were co-electroporated with siRNA1 and the 480-17Q or 480-68Q plasmids. For FK506 treatment, cells were treated with FK506 (0.1, 0.3, 1 μM; Alexis, Lausen, Switzerland) or vehicle (DMSO) for 30 min before videomicroscopy. All the experiments and in particular the videoexperiments were conducted in conditions in which no overt toxicity of the various constructs nor aggregation could be detected. The co-expression of BDNF and the various constructs as well as RNAi efficiency was verified by immunostaining after the videomicroscopy experiments. 95% or more of co-expression was observed. Single cell analysis of immunostaining levels revealed no difference in huntingtin expression between the different constructs nor signs of apoptosis in the conditions used.
Videomicroscopy experiments and imaging treatment
Videomicroscopy experiments were done 2-3 days after transfection. Cells were cotransfected with BDNF-mCherry and various constructs of huntingtin or the corresponding empty vectors with a DNA ratio of 1:4. Live videomicroscopy was carried out using a Leica DM IRBE microscope and a PL APO oil 100× objective with a numerical aperture of 1.40-0.70, coupled to a piezo device (PI) and recorded with Photometrics CoolSNAP HQ2 camera (Roper Scientific, Trenton, NJ) controlled by Metamorph software (Molecular Devices, Sunnyvale, CA). Stacks were acquired in cultured medium at 37°C for cortical neurons. Images were collected in stream set at 2 × 2 binning with an exposure time of 100 ms (frequency of 2s) with a Z-step of 300 nm. All stacks were treated by automatic batch deconvolution using the PSF of the optical system, Meinel algorithm with parameters set at 7 iterations, 0.7 sigma and 4 frequency. Maximal z and time projection, animations and analyses of vesicles tracking were done with ImageJ software as previously described . Supplemental movies and kymographs were obtained by collecting images at a frequency of 1 image/s with an acquisition time of 300 ms. The kymostacks were generated using a homemade ImageJ KymoToolbox plugin http://rsb.info.nih.gov/ij/, NIH, USA; available on request at Fabrice.Cordelieres@curie.fr). For image analyses of fixed samples, images were acquired at room temperature with a Leica DM RXA microscope with a PL APO oil 100× NA of 1.4 objective coupled to a piezo device (PI) and a Micromax RTE/CCD-1300-Y/HS camera controlled by Metamorph software. The mounting medium was 0.1 g/ml Mowiol 4-88 (Calbiochem, Merck Biosciences, Darmstadt, Germany) in 20% glycerol. Z-stack was of 200 nm. Deconvolution was performed as for videomicroscopy.
Western blot analysis
For neuronal cultures, neurons were washed with ice-cold PBS before scraping and lysis. In the case of experiments assessing the efficient silencing of huntingtin or calcineurin, half of the coverslips were lysed after videomicroscopy. For analyses of calcineurin in different brain regions, dissected frozen samples were directly resuspended in lysis buffer. Composition of buffers and all the procedures were performed as previously described . 6% and 10% acrylamide gels were respectively used for huntingtin and calcineurin detection. 8% acrylamide gels were used to detect the two proteins on the same blot. Membranes were blocked in 5%BSA/TBST buffer (20 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween 20) and immunoblotted with anti-CaN Pan A (1:1000; Chemicon) or anti-α-tubulin (1:10000; DM1A; Sigma, St Louis, MO), p150Glued (clone 1, BD Biosciences, San Jose, CA, USA), home made anti-phospho-htt-S421-714  and anti-huntingtin antibody mAb 4C8 (1:5000; clone 1HU-4C8, ) antibodies for 1 h. Membranes were then labelled with secondary IgG/HRP antibodies (Jackson ImmunoResearch, WestGrove, PA, USA), washed and incubated for 2 min with SuperSignalWest Pico Chemiluminescent Substrate (Pierce, Erembodegem, Belgium) according to the instructions of the supplier. Membranes were exposed to Kodak (Rochester, NY) BioMax films and then developed. Quantification of the signal was performed by densitometric scanning of the film using GelPRO analyzer software.
To ensure efficient silencing in electroporated neurons used for videomicroscopy, coverslips were fixed after videorecording with methanol for 3 min, blocked 1 h in PBS 1% BSA and incubated with mAb 4C8 anti-huntingtin antibody (1:100; ) during 90 min. Coverslips were rinsed three times in PBS 1:1000 tween 20 and incubated with a secondary Alexa 488 fluorescent antibody (Invitrogen, Oregon, USA). After incubation with DAPI (1:10000 in PBS; Roche, Indianapolis, USA), coverslips were rinsed three times and mounted with Mowiol.
Calcineurin activity was measured in primary cultures of cortical neurons from HdhQ111/Q111 mice, and in samples obtained from the cortex of 1 year old wild-type Hdh+/+ and mutant HdhQ111/Q111 and HdhQ111/+ mice using the Calcineurin Cellular Activity Assay Kit (Calbiochem, San Diego, CA, USA). For experiments on cultures, neurons were treated with FK506 or DMSO and then lyzed in the buffer supplied by the manufacturer. For cortex analyzes, samples obtained from brain dissection were homogeneized in the supplied buffer. For both experiments, samples were processed according to the protocol provided by the manufacturer. Calcineurin activity was determined as the difference between total phosphatase activities minus the phosphatase activity in presence of 10 mM EGTA that blocks calcineurin activity. Data were expressed as the percentage of the total phosphatase activity.
- The abbreviations used are DIV:
days in vitro
brain-derived neurotrophic factor
We acknowledge G. Grange for help with experiments; G. Banker for BDNF-mCherry; F.P. Cordelières and the Institut Curie Imaging Facility for image acquisition and treatment and members of the Saudou/Humbert's laboratory for helpful comments. This work was supported by grants from Agence Nationale pour la Recherche (ANR-MRAR-018-01 to FS and ANR-08-MNP-039 to FS), Fondation pour la Recherche Médicale (FRM) and Fondation BNP Paribas (F.S.) and, CHDI Inc. Foundation (RecID1766 to FS and SH). JRP was supported by fundación FECYT, RP by "Beatriu de Pinós" from Generalitat de Catalunya and CHDI Inc. Foundation and D.Z. by CHDI Inc. Foundation. FS and SH are Institut National de la Santé et de la Recherche Médicale/Assistance Publique-Hôpitaux de Paris investigators.
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