Background

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 [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 of neurons in the brain or disease progression. Promising treatment strategies involve the identification of compounds 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 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 [16]. 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 [15]. Consistent with this hypothesis, calcineurin levels have been found to be higher than normal in neuronal cells immortalized from HD mice [15]. 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 [15] and anti-huntingtin antibody mAb 4C8 (1:5000; clone 1HU-4C8, [49]) 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.

Immunofluorescence

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; [49]) 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

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.