Patent Application
20080300178
The present invention relates to methods for treating Huntington's disease, pharmaceutical compositions useful in such methods, and screening methods for identifying a compound useful for treating Huntington's disease.
Huntington's disease (HD) results from genetically programmed degeneration of neurons, in certain areas of the brain. The prevalence of the of HD in occidental world is 1 out of 10 000 people. This degeneration causes uncontrolled movements, loss of intellectual faculties, and emotional disturbance. HD is an autosomal dominant neurodegenerative disease. A person who inherits the HD gene will sooner or later develop the disease. Some early symptoms of HD are mood swings, depression, irritability or trouble driving, learning new things, remembering a fact, or making a decision. As the disease progresses, concentration on intellectual tasks becomes increasingly difficult and the patient may have difficulty feeding himself or herself and swallowing. The rate of disease progression and the age of onset vary from person to person. A genetic test, coupled with a complete medical history and neurological and laboratory tests, help physician's diagnose HD. Presymptomic testing is available for individuals who are at risk for carrying the HD gene. In 1 to 3 percent of individuals with HD, no family history of HD can be found.
At this time, there is no way to stop or reverse the course of HD. Drugs used to treat HD only alleviate symptoms and have side effects such as fatigue, restlessness, or hyperexcitability. Therefore, there is a strong need for HD treatment.
HD is caused by an abnormally expanded CAG tract in the coding region of the IT15 gene located in chromosome 4p16.3, which encodes a polyglutamine tract in the protein huntingtin (htt). Huntingtin becomes toxic when it contains an abnormal polyQ expansion. PolyQ-huntingtin is cleaved in the cytoplasm, and thereafter translocates to the nucleus where it forms ubiquitin immunopositive nuclear aggregates. When in the nucleus, polyQ-huntingtin induces transcriptional dysregulation and neuronal death through a gain of function mechanism (Ross, 2002;). A loss of the protective functions of huntingtin could act concomitantly and/or synergistically with the gain of new toxic functions (Cattaneo et al., 2001). In agreement, dysregulation of BDNF transcription is linked to the loss of huntingtin normal function ( ). In addition, when huntingtin contains the polyQ expansion, its ability to transport BDNF-containing vesicles and to promote neuronal survival is lost (Gauthier et al., 2004). Finally, aggregates are also found in neurites and could also participate in neuronal dysfunction by altering the microtubules network dynamics and/or axonal transport Charrin et al., 2005).
Several post-translational modifications such as proteolysis, ubiquitination and sumoylation modify the toxicity of huntingtin. (Another post-translational modification that plays an important role in disease is phosphorylation. In particular, the Ser/Thr kinases, Akt and the Serum and Glucocorticoid-induced Kinase, SGK, phosphorylate huntingtin at serine 421 (S421) (Humbert et al., 2002; Rangone et al., 2004; Warby et al., 2005). Phosphorylation at S421 subsequently abolishes polyQ-huntingtin induced toxicity in a cellular model of HD (Humbert et al., 2002; Rangone et al., 2004). However, it is not clear whether phosphorylation of S421 is crucial in vivo. In addition, huntingtin is phosphorylated at serine 434 by the cyclin-dependent kinase Cdk5 and this reduces its cleavage by caspases (Luo et al., 2005).
Immunophillin ligands, such as FK506 and cyclosporin, have been recently described as a putative therapeutic strategy for neuroregeneration and neuroprotection (Klettner and Herdegen, 2003; Pong and Zaleska, 2003). Therefore, immunophillin ligands have been suggested for treating neurodegenerative diseases. The immunophillin ligands have two kind of activity: 1) they inhibit peptidyl-prolyl isomerase (PPI or rotamase); 2) they inhibit calcineurin, thereby having an immunosuppressive function. However, the role of these two kind of activity in neuroprotection and neuroregeneration is still not clear.
Calcineurin is involved in the activation of neuronal nitric oxide synthase (nNOS). nNOS produces nitric oxide (NO) which leads to structural damage to lipids, proteins, and DNA. Otherwise, calcineurin dephosphorylates BAD and enhances its hetero-dimerization with Bcl-X1, leading to apoptosis. Therefore, inhibition of calcineurin can block the downstream mediators of cell death. Similarly, stabilization of mitochondrial function can also mediate neuroprotection.
Non-immunosuppressant immunophilin ligands (e.g., GPI-1046 and V10,367), devoid of calcineurin inhibition activity, have been developed for treating neurological disorders. In particular, clinical trials with GPI-1046 and V10,367 for treating Parkinson's disease have failed.
No specific data for HD treatment has been reported except Almeida et al (2004). This article reports an in vitro study on cortical cells treated by 3NP (3-nitropropionic acid) (a chemical model of HD which does not reproduce huntingtin mutation). 3NP treatment induces cell death and this cell death can be blocked by FK506 through an unclear mechanism not directly linked to the phosphoryaltion of huntingtin at positions S421.
The inventors demonstrate that phosphorylation of huntingtin at position S421 is neuroprotective in vivo and that calcineurin (CaN) dephosphorylates S421. Inhibition of CaN activity leads to an increased phosphorylation of huntingtin at S421 and prevents polyQ-huntingtin-induced death of neurons. Consequently, the inventors demonstrate the interest of a drug increasing the phosphorylation of huntingtin at position S421 as a therapeutic approach to treat HD by decreasing the polyQ-huntingtin-induced toxicity.
Therefore, the present invention concerns the use of a drug increasing the phosphorylation of huntingtin at position S421 for the manufacture of a medicament for blocking or reducing the toxicity of polyQ-huntingtin in a subject having Huntington's disease. In particular, the drug is aiming to block or reduce the neuronal death induced by polyQ-huntingtin. The drug increasing the phosphorylation of huntingtin at position S421 can be a drug increasing the activity of the kinase Akt, protein kinase A, Polo kinase 1, AuroraA and AuroraB and/or SGK. In a preferred embodiment, the drug increasing the phosphorylation of huntingtin at position S421 is a drug inhibiting the dephosphorylation of huntingtin at position S421. More preferably, the drug inhibiting the dephosphorylation of huntingtin at position S421 is a calcineurin inhibitor or a drug inhibiting the interaction between calcineurin and huntingtin or a dominant interfering form of calcineurin. In the most preferred embodiment, the drug inhibiting the dephosphorylation of huntingtin at position S421 is a calcineurin inhibitor. In one embodiment, the calcineurin inhibitor is selected from the group consisting of cyclosporin A, FK506, FK520, L685,818, FK523, 15-0-DeMe-FK-520, Lie120, fenvalerate, resmethrin, cypermethrin, deltamethrin and an analogue thereof. Preferably, the calcineurin inhibitor is selected from the group consisting of FK506, cypermethrin, deltamethrin and an analogue thereof. More preferably, the calcineurin inhibitor is FK506. Alternatively, the calcineurin inhibitor can be a RNA interference specific of calcineurin. The subject having Huntington's disease carries the Huntington's disease mutation. In particular, the HD mutation is an abnormal expansion (superior to 35) of the CAG repeat in the coding region of the IT15 gene. This subject can be presymptomatic or can also have developed the symptoms of HD. In this case, the drug increasing the phosphorylation of huntingtin at position S421 can be used in combination with a drug alleviating symptoms of Huntington's disease.
In a most preferred embodiment, the present invention concerns the use of FK506 for the manufacture of a medicament for blocking or reducing the toxicity of polyQ-huntingtin in a subject having Huntington's disease by inhibiting the dephosphorylation of huntingtin at position S421.
In a further embodiment, the present invention concerns a method for selecting, identifying or screening a compound useful for treating a subject having Huntington's diseases, comprising the selection or identification of a compound capable of increasing the phosphorylation of huntingtin at position S421.
Using a rat model of HD based on lentiviral-mediated expression of a polyQ-huntingtin fragment in the striatum, the inventors demonstrate that phosphorylation of S421 is neuroprotective in vivo. They also demonstrate that calcineurin (CaN), a calcium/calmodulin-regulated Ser/Thr protein phosphatase, dephosphorylates S421 in vitro and in cells. Importantly, inhibition of CaN activity either by overexpression of a dominant-interfering form, by RNA interference or by the specific inhibitor FK506, leads to an increased phosphorylation of huntingtin at S421 and prevents polyQ-mediated death of striatal neurons. Finally, the inventors show that administration of FK506 to mice increases huntingtin S421 phosphorylation in brain. Collectively, these data highlight the importance of CaN in the modulation of S421 phosphorylation and suggest the potential use of CaN inhibition as a therapeutic approach to treat HD as it decreases the polyQ-induced toxicity.
Calcineurin (CaN), also known as protein phosphatase 2B (PP2B), is a phosphoprotein Ser/Thr phosphatase activated physiologically by Ca2+-calmodulin (for a review see Mansuy, 2003). Therefore, it couples intracellular calcium to the dephosphorylation of selected substrates, which include transcription factors (Nuclear Factor of Activated T cells, NFAT), ion channels (inositol-1,4,5 triphosphate receptor), proteins involved in vesicular trafficking (amphyphysin, dynamin), scaffold proteins (AKAP79) and phosphatase inhibitors (DARPP-32, inhibitor-1). CaN is present in all tissues in mammals, with notably high levels in brain, some studies indicating that it may account for 1% of the total protein content of the brain. The catalytic subunit is mainly expressed in the cortex, striatum and the hippocampus. Within the central nervous system, CaN activity has been involved in synaptic plasticity, as it is considered a negative constraint for long-term memory. In nerve terminals, CaN triggers synaptic vesicle endocytosis by dephosphorylating vesicular and plasma membrane proteins in response to Ca2+. Finally, CaN is inhibited by drugs such as cyclosporin A and FK506 through the binding of these drugs to their appropriate receptors (immunophilins); Hamawy, 2003). FK506 and its derived immunophilin ligands, unlike cyclosporin, can readily cross the blood-brain barrier (Pong and Zaleska, 2003).
The inventors have previously shown that huntingtin is a processivity factor for intracellular transport of vesicles such as Brain Derived Neurotrophic factor (BDNF). In HD, the transport of BDNF vesicles in cortical neurons is altered leading to a reduction of trophic support and neuronal toxicity in the striatum. In the present invention, the inventors demonstrate that huntingtin phosphorylation at S421 by the IGF-1/Akt pathway rescues transport altered by the polyQ expansion and, by promoting an outward BDNF flow increases neurotrophic support. These results elucidate the neuroprotective effect of S421 phosphorylation in HD and suggest that drugs enhancing huntingtin phosphorylation are of therapeutic interest since phosphorylation of S421 rescues huntingtin function in transport. In addition, the inventors demonstrate that huntingtin phosphorylation by Akt at serine 421 regulates the ability of polyQ-huntingtin but also normal huntingtin to promote transport.
The present invention concerns the use of a drug increasing the phosphorylation of huntingtin at position S421 for the manufacture of a medicament for blocking or reducing the toxicity of polyQ-huntingtin in a subject having Huntington's disease. Preferably, the drug is aiming to block or reduce the neuronal death induced by polyQ-huntingtin. The drug is also aiming to restore the huntingtin's activity in the axonal transport.
The present invention also concerns a method for treating a subject having Huntington's disease by administering a therapeutic amount of a drug increasing the phosphorylation of huntingtin at position S421, thereby blocking or reducing the toxicity of polyQ-huntingtin. In particular, the drug is aiming to block or reduce the neuronal death induced by polyQ-huntingtin. The drug is also aiming to restore the huntingtin's activity in the axonal transport.
The subject having Huntington's disease is a subject that carries the HD mutation carrying a Huntington disease gene. The mutation that causes Huntington's disease is an anormal CAG repeat expansion in the huntingtin gene (also called IT15 gene). Indeed, in normal individuals, the repeat occurs between 6 and 35 times. In a subject having HD, the repeat occurs more than 36 times, generally from 40 times to more than 80 times. This mutation results in a polyglutamine (poly-Q) expansion in the protein named huntingtin.
The subject having Huntington's disease can be presymptomatic. The presymptomatic subject is identified by a genetic test. U.S. Pat. No. 4,666,828 discloses a method for detecting the presence in a subject of the gene for Huntington's disease comprising the analysis of a DNA polymorphism linked to Huntington's Disease on the human chromosome 4, in particular a RFLP. In a preferred embodiment, the test analyzes DNA directly for the presence of the Huntington's disease mutation (Kremer et al, 1994). Of course, any test allowing to determine if a subject has an anormal huntingtin protein or gene is contemplated in the present invention.
Accordingly, the present invention concerns a method for treating HD subject comprising: detecting the presence in the subject of the HD mutation; and, administering a therapeutic amount of a drug increasing the phosphorylation of huntingtin at position S421 to the subject having the HD mutation, thereby blocking or reducing the neuronal death induced by polyQ-huntingtin. In particular, detecting the presence in the subject of the HD mutation comprises the determination of the number of CAG repeat in the IT15 gene. Subject having more than 36 repeats, preferably more than 40 repeats, is considered as having the HD mutation. Alternatively, detecting the presence in the subject of the HD mutation can also be done at the huntingtin protein level by determining the size of the polyglutamine expansion.
Alternatively, the subject having Huntington's disease can have HD symptoms. HD symptoms are well-known by the one skilled in the art. For instance, they comprise uncontrolled movements, loss of intellectual faculties (e.g., dementia), and emotional disturbance (e.g., depression). In this case, the drug increasing the phosphorylation of huntingtin at position S421 can be used in combination with a drug alleviating symptoms of Huntington's disease. For example, such drugs treating HD symptoms can be antipsychotics (haloperidol, chlorpromazine, olanzapine), antidepressants (fluoxetine, sertraline hydrochloride, nortriptyline), tranquilizers (benzodiazepines, paroxetine, venlafaxin, beta-blockers), mood-stabilizers (lithium, valproate, carbamazepine), and Botulinum toxin.
Preferably, the subject is a primate, more preferably a higher primate, most preferably a human.
The treatment of a subject having HD can result in an improved motor coordination, an increased survival, the prevention of Huntington's disease from occurring in a presymptomatic HD subject, the retardation or arrest of HD development and/or a regression of HD. Indeed, the treatment according to the present invention results in blocking or reducing the toxicity of polyQ-huntingtin, thereby blocking or reducing the neuronal death induced by polyQ-huntingtin. Therefore, at an early stage of HD such as presymptomatic stage, the HD development should be prevented. At a later stage of HD, the HD development should be inhibiting or slowing down. The inventors also showed in the present invention that the increased phosphorylation of huntingtin at S421 results in the full or partial recovery of its activity in the axonal transport, in particular BDNF transport which is linked to the anti-apoptotic action of normal huntingtin.
Due to the increased anti-neuronal death role of huntingtin following the treatment according to the present invention through its increased axonal transport capacity, the present invention further contemplates the use of a drug increasing the phosphorylation of huntingtin at position S421 for the manufacture of a medicament for increasing the axonal transport by huntingtin in a subject suffering from or at risk of a neurodegenerative disease. The neurodegenerative disease can be selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The present invention also contemplates a method for treating a subject suffering from or at risk of a neurodegenerative disease comprising administering a therapeutic amount of a drug increasing the phosphorylation of huntingtin at position S421, thereby increasing the axonal transport by huntingtin. The increased axonal transport by huntingtin is aiming to have an anti-apoptotic effect.
A drug increasing the phosphorylation of huntingtin at position S421 can be a drug increasing the activity of the kinase Akt, protein kinase A, Polo kinase 1, AuroraA, AuroraB and/or SGK. However, in a preferred embodiment, the drug increasing the phosphorylation of huntingtin at position S421 is a drug inhibiting the dephosphorylation of huntingtin at position S421. More preferably, the drug inhibiting the dephosphorylation of huntingtin at position S421 is a calcineurin inhibitor or a drug inhibiting the interaction between calcineurin and huntingtin. In the most preferred embodiment, the drug inhibiting the dephosphorylation of huntingtin at position S421 is a calcineurin inhibitor.
Calcineurin inhibitors include, but are not limited thereto, cyclosporin A (Novartis International AG, Switzerland), FK506 (Fujisawa Healthcare, Inc., Deerfield, Ill., USA), FK520 (Merck & Co, Rathway, N.J., USA), L685,818 (Merck & Co), FK523, 15-0-DeMe-FK-520 (Liu, Biochemistry, 31:3896-3902 (1992)), Lie120, fenvalerate (Merck & Co), resmethrin (Merck & Co), cypermethrin (Merck & Co) and deltamethrin (Merck & Co). WO2005087798 describes cyclosporine derivative inhibiting calcineurin.
Calcineurin is a heterodimer composed of a catalytic subunit (Calcineurin A) and a regulator subunit (Calcineurin B). Then, the activity of calcineurin can also be inhibited by blocking its expression, in particular the expression of one of its subunit. In a preferred embodiment, the activity of calcineurin can also be inhibited by blocking the expression of the regulator subunit B. In an alternative preferred embodiment, the activity of calcineurin can also be inhibited by blocking the expression of the regulator subunit A, either CaNAα or CaNAβ. The expression can be blocked by any mean known by one skilled in the art, e.g., by chemically synthesized oligonucleotides such as antisense oligonucleotides, ribozymes, short interfering RNA (siRNA) and short hairpin RNA (shRNA). Antisense oligonucleotides are short single-strand molecules that are complementary to the target mRNA and typically have 10-50 mers in length, preferably 15-30 mers in length, more preferably 18-20 mers in length. Antisense oligonucleotides are preferably designed to target the initiator codons, the transcriptional start site of the targeted gene or the intron-exon junctions (for review, Kurreck, 2003). Ribozymes are single stranded RNA molecules retaining catalytic activities. The mechanism of ribozyme action involves sequence specific interaction of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The ribozyme is engineered to interact with the target RNA of interest comprising a cleavage NUH triplet, preferentially GUC (for review, Usman et al., 2000). siRNA are usually 21 or 23 nucleotides long, with a 19 or 21 nucleotides duplex sequence and 2 nucleotides-long 3′ overhangs. shRNA are designed with the same rules than for a sequence encoding a siRNA excepting several additional nucleotides forming a loop between the two strands of the siRNA. (For review, see Mittal, 2004). In a particular embodiment, the inhibiting oligonucletide is expressed by a vector, preferably a viral vector comprising a contruct allowing the expression of inhibiting oligonucleotides. For instance, the viral vector can be an adenovirus, an AAV, a lentivirus or a HSV. Examples disclosed in detailed one way to performed the calcineurin inhibition by siRNA.
In a particular embodiment, the present invention concerns a RNA interference specific of calcineurin as a medicament. In a preferred embodiment, the present invention concerns the use of a RNA interference specific of calcineurin for the manufacture of a medicament for treating Huntington's disease. Such a RNA interference is directed against CaNAα (NM—000944) and/or CaNAβ (NM—021132).
Calcineurin inhibitor can also be a dominant-interfering form of calcineurin, in particular of CaNA and/or of CaNB. The dominant-interfering form of calcineurin can be for example CaNA-D130N. Other examples of dominant-interfering form of calcineurin are CaNA-H101Q (Wang et al, 1999, Science 284, 339-343), CaNA-H160Q and CaNA-H290Q (Shibasaki et al, 1996, Nature, 6589, 370-373; Nishimura and Tanaka, 2001, J. Biol. Chem., 276, 19921-19928), H160Q (Zhu et al, 2000n J. Biol. Chem., 275, 15239-15245), D148-152 (Yamashita, 2000, J. Exp. Med., 191, 1869-1880) and Dnter-DcaM (muramatsu and Kincaid, 1996, BBRC, 218, 466-472; Musaro et al, 1999, Nature, 6744, 581-585). Therefore, the dominant-interfering form of calcineurin is expressed by a vector, preferably a viral vector comprising a contruct allowing its expression. For instance, the viral vector can be an adenovirus, an AAV, a lentivirus or a HSV.
Alternatively, the activity of calcineurin can be inhibited by a drug that inhibits the interaction between the calcineurin subunits, in particular the interaction between subunits A and B. The activity of calcineurin can be inhibited by a drug that inhibits the interaction between the calcineurin and calmodulin. Calcineurin inhibition can also be obtained by activation of endogenous inhibitors of calcineurin, including cabin1, calcipressins and AKAP79.
By “analogue” of a drug is intended a compound having similar structural features and having the same biological activity, in particular which inhibits calcineurin.
Other drugs inhibiting the calcineurin can be identified by screening methods already disclosed in the art. As illustration, the U.S. Pat. Nos. 6,875,581 and 6,338,946 describe screening methods useful for identifying modulators of calcineurin activity.
Therefore, the present invention also concerns methods for selecting, identifying or screening a compound useful for treating a subject having Huntington's diseases, comprising the selection or identification of a compound capable of increasing the phosphorylation of huntingtin at position S421.
The method can comprise:
Alternatively, the method can comprise:
In a preferred embodiment, the phosphorylation of huntingtin at position S421 is detected with a antibody specific huntingtin phosphorylated at position S421, in particular by Western Blot. Preferably, the cell is a neuron, in particular a stratial neuron. However, any kind of cell is contemplated in the present invention.
The drug inhibiting the calcineurin may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., in isolated or in mixture with other substances. In a preferred embodiment, the drug is a small molecule. In an other preferred embodiment, the drug is a peptide or a polypeptide. In an additional embodiment, the drug is a nucleic acid, e.g., an antisense, a siRNA, a ribozyme. In another embodiment, the drug is an aptamer. In a preferred embodiment, the drug inhibiting calcineurin crosses the blood-brain barrier.
The drug used in the present invention can be formulated as a pharmaceutical composition generally comprising a pharmaceutically acceptable carrier. By a pharmaceutically acceptable carrier is intended a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the drug with which it is administered. For example, a pharmaceutically acceptable carrier can be physiological saline solution. Other pharmaceutically acceptable carriers arc known to one skilled in the art and described for instance in Remington: The Science and Practice of Pharmacy (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins).
The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. For drugs that do not pass the blood-brain barrier, the drugs need to be injected into the brain, for example by a stereotaxic injection.
By a “therapeutic amount” is intended an amount of a drug that is sufficient to increase the phosphorylation of huntingtin at position S421. The effective amount of a drug varies depending upon the administration mode, the age, body weight, sex and general health of the subject. In a preferred embodiment, it is an amount that is sufficient to effectively inhibit the calcineurin activity, thereby blocking or reducing the polyQ-huntingtin toxicity. The effect can be assessed by the phosphorylation state of huntingtin at position S421, through the neuronal death induced by polyQ-huntingtin. It will be appreciated that there will be many ways known in the art to determine the therapeutic amount for a given application. For instance, the dose of FK506 can be from 0.001.mg/kg/day to 10 mg/kg/day, preferably between 0.01 and 10 mg/kg/day by oral administration and between 0.001 and 1 mg/kg/day by intravenous injection, preferably between 0.01 and 0.5 mg/kg/day. The administration protocols are well-known by one skilled in the art. In a particular embodiment, the blood FK506 level is comprised between 5 and 40 ng/ml, preferably between 15 and 20 ng/ml. Accordingly, the administered dose of FK506 can be adapted in order to obtain the above-mentioned blood FK506 level.
The following example illustrates the invention.
Constructs. The pSIN-480-17Q, pSIN-480-68Q, pSIN-480-68Q-S421A and pSIN-480-68Q-S421D were generated from the 480-17Q, 480-68Q, 480-68Q-S421A, and 480-68Q-S421D plasmids respectively (Humbert et al., 2002). These plasmids encode the first 480 amino acids fragment of huntingtin with 17 or 68 glutamines and a serine to alanine mutation (S421A) or a serine to aspartic acid mutation (S421D) at position 421. First, a PCR strategy was used to modify the C-terminal part of the 480 constructs (QuickChange site-directed mutagenesis; Stratagene, La Jolla, Calif., USA) using the forward primer: 5′ GCAGCTCACTCTGGTTCAAGAAGAG 3′ (SEQ ID No 1) and the reverse primer:
5′CTCGAGTTAAGCGTAATCTGGAACATCGTATGGGTAGGATCTAGGC TGCTCAGTG 3′ (SEQ ID No 2) containing a hemmaglutinin-tag (HA). The various fragments were cloned into the parental 480-17Q/68Q plasmids resulting in the generation of the vectors 480-17Q/68Q with S421, S421A and S421D mutation with a C-terminal HA tag and then subcloned in the SINW-PGK-GDNF (BamHI-XhoI; Deglon et al., 2000).
The lentiviral particles were produced in 293T cells and resuspended in phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) as previously reported). The particle content of viral batches was determined by p24 antigen ELISA (PerkinElmer Life Sciences, Boston, Mass., USA).
The vectors encoding wild-type Akt, constitutively active Akt (Akt c.a.), wild-type CaNA, a constitutively-active (Ca2+-insensitive) form of CaNA (CaNA-ΔCaM), a catalytic-dead dominant interfering form of CaNA (CaNA-D130N) and CaNB, BDNF-eGFP, and the various huntingtin constructs 480-17Q, 480-68Q, 480-17Q-S421A, 480-68Q-S421A, 480-17Q-S421D, 480-68Q-S421D, WT and polyQ-FL-htt have been already described.
Animals. Adult female Wistar rats (Iffa-Credo/Charles River, Les Oncins, France) weighting about 180-200 g were used. The animals were housed in a controlled temperature room that was maintained on a 12 h light/dark cycle. Food and water were available ad libitum.
FK506 (Alexis, Lausen, Switzerland) was administered to C57/BL6 male mice aged 5-6 weeks (Iffa-Credo/Charles River, Les Oncins, France) orally or by intra-peritoneal injections (5 mg/kg) (Dunn et al., 1999; Singh et al., 2003). For oral administration, FK506 was dissolved in 100 μl 0.5% carboxymethylcellulose (Sigma). For intraperitoneal injections, FK506 was dissolved in 200 μl of Cremophor® (Sigma). Mice were sacrificed at indicated times post administration. The whole brain was homogenized in 1% Triton X-100 lysis buffer (see Western blot analysis section) supplemented with 1 μM okadaic acid (Sigma), 1 μM FK506 (Alexis), 1 μM cyclosporine A (Sigma) and 40 nM tautomycin (Calbiochem, Darmstadt, Germany) and centrifuged at 20,000×g (5 min; 4° C.). An aliquot of the supernatant was resolved by 6% SDS-PAGE.
Studies using animals were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health, USA.
Injection of the lentiviruses. Concentrated viral stocks were thawed and resuspended by repeated pipetting. Lentiviral vectors were stereotaxically injected (David Kopf Instruments, Tujunga, Calif., USA) into the striatum of ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthetized animals using an Hamilton syringe with a 34-gauge blunt-tip needle (Hamilton, Reno, Nev., USA). For each vector, particle content was matched to 200,000 ng p24/ml. The viral suspensions (4 μl) were injected at 0.2 μl/min by means of an automatic injector (Stoelting Co., Wood Dale, Ill., USA) and the needle was left in place for an additional 5 min. The stereotaxic coordinates were: 0.5 mm rostral to bregma; 3 mm lateral to midline and 5 mm from the skull surface. The skin was closed using a 6-0 Prolene® suture (Ethicon, Johnson and Johnson, Brussels, Belgium).
Histological processing. 1, 12 and 24 weeks after lentiviral injection, the animals were given a sodium pentobarbital overdose and transcardially perfused with saline and 4% paraformaldehyde/10% picric acid. The brains were removed and postfixed in 4% paraformaldehyde/10% picric acid for approximately 24 h and finally cryoprotected in 25% sucrose/0.1 M phosphate buffer for 48 h. The brains were frozen in dry ice and 25 μm coronal sections were cut on a sliding microtome cryostat (Cryocut 1800, Leica Microsystems AG, Glattbrugg, Switzerland) at −20° C. Slices throughout the entire striatum were collected and stored in 48 well trays (Costar, Cambridge, Mass., USA) as free floating sections in PBS supplemented with 0.12 μM sodium azide. The trays were stored at 4° C. until immunohistochemical processing.
Striatal sections from injected rats were processed by immunochemistry for dopamine and cAMP-regulated phosphoprotein of a molecular mass of 32 kDa (DARPP-32; 1:7500; Chemicon International Inc., Temecula, Calif., USA), and for HA-tag (monoclonal anti-HA antibody; 1:1000; Covance Research Products, Berkeley Calif., USA) as previously described (Bensadoun et al., 2001). Sections were subsequently incubated with the biotinylated secondary goat anti-mouse or goat anti-rabbit antibodies (Vector laboratories, Burlingame, Calif., USA) and the visualization was done as previously described
Quantification of the DARPP-32 depleted regions and statistical analyses. The downregulation of DARPP-32 was measured with an image analysis program (NIH Image 1.63) in each section containing the lesion. For each section, the optical density is first determined in the targeted region, the striatum, to obtain the T value and, in a non infected region of the striatum (NI) that did not received the virus. A background value is then obtained from a DARPP-32-non expressing region (B, cortex). The percentage of lesion is then calculated using the following formula: % of lesion=100−((N1−B)/(T−B)×100) and is expressed as a percentage of lesion; 100% lesion corresponding to the lesion induced by the 480-68Q construct. Comparisons between two constructs injected in the same animal were made using paired t-tests. At 24 weeks, the striatal volume is not modified by the various lentiviruses (data not shown).
In vitro phosphorylation/dephosphorylation assays. Kinase assays were performed as described (Rangone et al., 2004) using recombinant Akt (Upstate Biotechnology, Charlottesville, USA) and a purified truncated form of huntingtin protein as a substrate (aminoacids 384 to 467 of human huntingtin fused with GST; 1 hr incubation, 30° C.). Purified calcineurin (native protein isolated from bovine brain, Upstate Biotechnology, Temecula, Calif., USA) was added as stated. The reaction products were resolved by 12% SDS-PAGE.
Cell culture, transfection and drug treatments. Primary cultures of striatal neurons were prepared from E17 Sprague Dawley rats and transfected at 4 days in vitro by a modified calcium phosphate technique (Saudou et al., 1998). Mouse neuronal cells derived from wildtype huntingtin mice (neuronal cells, +/+) and from HdhQ109 knock-in mice (109Q/109Q) were cultured as previously described (Trettel et al., 2000) and transfected with Lipofectamine 2000 (Invitrogen, Breda, The Netherlands). When cotransfected (
Western blot analysis. After transfection/incubation with drugs, cells were washed with ice cold phosphate-buffered saline prior to scraping and lysis. Lysis buffer consisted on 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (v/v) β-mercaptoethanol, 250 μM PMSF, 10 mg/ml aprotinin and leupeptin. Cell lysates were centrifuged at 20,000×g for 5 min at 4° C. Equal amounts of protein (40 μg) were subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes (Immobilon-P, Millipore). Blots were blocked in 5% bovine serum albumin (BSA)/TBST buffer (20 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween-20) and immunoblotted with anti-α-tubulin (1:1000; DM1A, Sigma), anti-calcineurin Pan A (1:1000; Chemicon), anti-phosphohuntingtin-S421-763 (Humbert et al., 2002), anti-phospho-huntingtin-S421-714 (see below), anti-huntingtin 1259 (1:1000)) and MAB2166 (1:5000; Chemicon) antibodies for 1 hr. Blots were then labelled with anti-rabbit IgG:HRP (Jackson ImmunoResearch, West Grove, USA), washed and incubated for 5 min with SuperSignal WestPico Chemiluminescent Substrate (Pierce, Erembodegem, Belgium) according to the supplier's instructions. Membranes were exposed to Kodak Biomax films and developed. Quantification of the signal was performed by densitometric scanning of the film using ImageJ software.
Immunofluorescence experiments. SHSY-5Y cells were grown on uncoated glass coverslips, transfected with wild-type CaNA/B for 24 hours and treated for 15 min with 1 μM ionomycin (Sigma) or left untreated. Cells were fixed with 4% paraformaldehyde-PHEM buffer for 20 min (in mM: PIPES, 120; HEPES, 50, EGTA, 20; Mg-acetate 4) and incubated with antiphospho-S421-huntingtin-763 (1:100) and anti-CaNA (1:200; Cat#C1956, Sigma) antibodies. Pictures were captured with a three-dimensional deconvolution imaging system as previously described (Gauthier et al., 2004). Mean fluorescence intensity of the phospho-S421 signal was next quantified using Metamorph software (Universal Imaging Corp, Princeton, N.J., USA). For each pair of cells (transfected/non transfected), signal from the transfected cell was standardized by giving a value of 100 to the non-transfected cell. Only non-mitotic cells of the same approximate size were considered. More than 30 cells in total were analyzed in two independent experiments.
Neurons were grown on laminin and polyD-lysine-coated glass coverslips, fixed as described before and incubated with the following primary antibodies: anti-phospho-S421-huntingtin-714 (1:100), N-ter 1259 (1/500) and CaNA (1:200). Secondary antibodies were anti-mouse AlexaFluor-488 (1:200) and anti-rabbit AlexaFluor-555 (1:200; Molecular Probes, Eugene, Oreg., USA). Pictures were captured with a three-dimensional deconvolution imaging system.
Measurement of neuronal survival. Four days post-plating, primary cultures of striatal neurons were transfected with wild-type or polyQ-huntingtin and green fluorescent protein (GFP) to identify the transfected cells. To be certain that each neuron synthesizing GFP also expressed the huntingtin construct, transfections were performed using a derived phosphate calcium method with a high ratio of huntingtin DNA to GFP DNA (10:1 ratio) (Humbert et al., 2002). Under these conditions over 95% of the GFP positive neurons also express the huntingtin construct (data not shown). GFP positive neurons were scored using fluorescence microscopy in a blinded manner 16 h and 36 h post transfection. Cell death occurring within the GFP positive cells was determined as the difference in the number of surviving neurons between the 2 time points and expressed as a fold increase in neuronal cell death relative to the death induced by the 480-17Q construct. Each graph represents two to four independent experiments performed in triplicate. Each bar in a given graph corresponds to the scoring of about 2000 neurons. For RNA interference experiment, neuronal cell death is expressed as the number of 480-68Q transfected cells that survived in presence of the various siRNA relative to the 480-68Q/scrambled condition. Data were submitted to complete statistical analysis.
siRNA against calcineurin. The siRNA sequences targeting rat CaNAα and CaNAβ correspond to the coding regions 677-695 (Acc. No. NM 017041) and 448-466 (Acc. No. NM 017042), respectively. More particularly, siRNA sequences targeting CaNAα are the following:
and siRNA sequences targeting CaNAβ are the following:
3 μg of siRNA or scrambled RNA were mixed with 4×106 freshly isolated striatal neurons, nucleofected following manufacturer's instructions (Amaxa Biosystems, Germany), plated onto 12 well plates and incubated for 40 hours. The scrambled RNA has the same nucleotide composition than the CaNAα siRNA but lacks a significant sequence homology to any other gene.
Antibodies against huntingtin phosphorylated on S421. Human specific anti-phosphohuntingtin-S421-763 antibody was previously described (Humbert et al., 2002). Generation of mouse specific anti-phospho-huntingtin-S421-714 antibody: phosphopeptide corresponding to mouse huntingtin sequence (CARGRSGS[PO3H2]IVELL) was synthesized, coupled to keyhole limpet hemocyanin (Neosystem, Strasbourg, France), and injected into rabbits. Polyclonal antibody was obtained from serum and affinity-purified with a phospho-peptide column. Briefly, the serum was filtered (0.22-μm filter), and following addition of 1 M Tris (pH 8.0) up to a final concentration of 100 mM, it was applied to a Sulfolink column (Pierce, Erembodegem, Belgium) coupled to the phosphorylated peptide. Retained antibodies were eluted with 100 mM glycine buffer (pH 2.7) and pH was rapidly neutralised with 1 M Tris pH 9. Antibodies were concentrated (Vivaspin concentrator 10000 MW, Viva Sience, Hannover, Germany) and stored in 50% glycerol.
Videomicroscopy Experiments and Analyses. For videoexperiments rat primary cortical neurons, +/+, and 109Q/109Q cells established from HdhQ111 knock-in mouse were prepared, cultured and transfected as previously described (Gauthier et al., 2004; Humbert et al., 2002; Saudou et al., 1998; Trettel et al., 2000). In some cases, primary cultures of cortical neurons are electroporated with the rat neuron Nucleofector® kit according to the supplier's manual (Amaxa, Cologne). Forskolin (10 M; Sigma) and IBMX (100 M; Sigma) were added to the cultures 5 hours after transfection.
Videomicroscopy experiments were done two or four days after transfection. Cells were co-transfected with BDNF-eGFP and various constructs of htt or the corresponding empty vectors with a DNA ratio of 1:4. Live videomicroscopy was carried out using an imaging system previously detailed (Gauthier et al., 2004). Cells were grown on glass coverslip that was mounted in a Ludin chamber. The microscope and the chamber were kept at 37° C. (33° C. for knock-in cells). Stacks of 10-15 images with a Z-step of 0.3 μm were acquired with a 100× PlanApo N.A. 1.4 oil immersion objective coupled to a piezo device (PI). Images were collected in stream mode using a Cool Snap HQ camera (Ropper Scientific) set at 2×2 binning with an exposure time of 50 to 100 ms (frequency of 2). All stacks were treated by automatic batch deconvolution using the PSF of the optical system. Projections, animations and analyses were generated using ImageJ software. Dynamics were characterized by tracking positions of eGFP vesicles in cells as a function of time with a special develop plug-in (http://rsb.info.nih.gov/ij/plugins/track/track.html). During tracking, the Cartesian coordinates of the centers of vesicles were used to calculate dynamic parameters (velocity, pausing time, directionality).
Phosphorylation of polyQ-Huntingtin at Serine 421 is Neuroprotective In Vivo
The inventors have previously demonstrated that the IGF1/Akt pathway is neuroprotective in a cellular model of HD (Humbert et al., 2002). Indeed upon IGF-1 activation, Akt phosphorylates polyQ-huntingtin at S421 and blocks its toxicity in primary cultures of striatal neurons. To examine whether phosphorylation at S421 plays a role in vivo and could therefore be a therapeutic target, the inventors used a rat model of HD based on lentiviral-mediated expression of polyQ-huntingtin in the striatum. This model recapitulates several features observed in HD patients such as the presence of neuritic and intranuclear inclusions, neuronal dysfunction and death. The inventors generated HA-tagged lentiviral constructs encoding the first 480 amino acids of huntingtin containing 17 (wild-type, 480-17Q) or 68 glutamine residues (mutant, 480-68Q) with either an intact S421, a S421 to alanine (S421A) or a S421 to acid aspartic (S421D) mutation. Lentiviruses were then injected into rat striatum to allow direct comparison for a given rat between the 480-17Q/480-68Q, 480-68Q/480-68Q-S421A and 480-68Q/480-68Q-S421D constructs (
Because phosphatase activities could allow a dynamic regulation of S421 phosphorylation, the inventors aimed to identify phosphatases that would dephosphorylate S421. Calcineurin, also known as protein phosphatase 2B (PP2B), is a calcium- and calmodulin-dependent phosphatase (for reviews see Mansuy, 2003). To assess whether calcineurin acts on S421, the inventors first performed dephosphorylation experiments in vitro. For this purpose, they used a polyclonal antibody that only binds to the phospho-serine at S421 (anti-phospho-huntingtin-S421-763) on the huntingtin protein (Humbert et al., 2002). In addition, they have previously generated a GST-fused form of huntingtin (GST-huntingtin, amino acids 384 to 467 of human huntingtin fused to GST) that is phosphorylated by Akt and SGK on S421 (Humbert et al., 2002). They incubated this huntingtin fragment with a constitutive active form of Akt (Akt c.a.) resulting in the phosphorylation of GST-huntingtin at S421 as detected with the anti-phospho-huntingtin-S421-763 antibody (
Calcineurin is a heterodimer composed of a 60 kDa catalytic subunit (CaNA) and a 19 kDA regulatory subunit (CaNB) (Rusnak and Mertz, 2000). Heterodimeric holoenzyme is necessary for its phosphatase activity. To determine whether calcineurin dephosphorylation of huntingtin at S421 occurs in cells, the inventors cotransfected immortalized mouse striatal cells (+/+ cells) with 480-17Q, a constitutive active form of calcineurin A (CaNA-ΔCaM), CaNB and/or Akt (
To test whether endogenous huntingtin is the target of calcineurin, the inventors used a dominant interfering form of CaNA where aspartic acid 130 is mutated into an asparagine (CaNA-D130N). Cotransfection of this construct with a NFAT reporter in HEK293 cells reduced endogenous NFAT activity by 60% (not shown). When CaNA-D130N encoding construct was transfected in the human neuroblastoma SHSY-5Y cell line, phosphorylation of endogenous huntingtin at S421 was increased (
To further demonstrate that calcineurin indeed dephosphorylates huntingtin at S421 in cells, the inventors transfected neuronal cells with wild-type CaNA/CaNB and analyzed phosphorylation of endogenous huntingtin by immunofluorescence using the anti-phospho-huntingtin-S421-763 antibody in control conditions or after activation of calcineurin by the calcium ionophore ionomycin (
Finally, the inventors confirmed the relevance of their findings by verifying that in their experimental system, calcineurin and huntingtin are present in the same striatal neurons. They prepared primary cultures of striatal neurons that were subsequently immunostained for CaNA, huntingtin and phospho-S421-huntingtin. They found that most if not all calcineurin immunopositive striatal cells were also immunoreactive for total and phosphorylated huntingtin at S421 (
The inventors have previously shown that phosphorylation of huntingtin at S421 is neuroprotective. They therefore investigated whether a dominant interfering form of calcineurin possesses neuroprotective properties by studying a neuronal model of HD that recapitulates the main features of the disease (
To further confirm the role of calcineurin inhibition on polyQ-huntingtin-induced cell death, the inventors decreased the levels of calcineurin by RNA interference. Two isoforms of calcineurin A subunits can be found in the brain, CaNAα and CaNAβ. The inventors therefore targeted both α and β subunits by RNA interference and found that the presence of both siRNA were necessary to ensure a significant decrease in the levels of CaNA (
Does the neuroprotection mediated by CaNA-D130N depend on S421? To answer this question, the inventors cotransfected CaNA-D130N in striatal neurons either with the 480-68Q construct or with a 480-68Q contruct where S421 has been mutated into an alanine, 480-68Q-S421A (
Therefore, calcineurin exerts its effect, at least in part, through the dephosphorylation of S421 on polyQ-huntingtin.
FK506 is an immunosuppressant drug used routinely in human treatment after transplantation. This compound mediates immunosuppression by inhibiting CaN-mediated dephosphorylation of NFAT, a transcription factor that regulates the expression of IL-2, which in turn regulates T-lymphocyte proliferation. Jurkat T cell line is a T-lymphocyte cell line extensively used to study calcium signaling pathways in which calcineurin participates ( ). To determine the effect of inhibiting calcineurin on the phosphorylation of huntingtin at S421, the inventors treated Jurkat T cells with FK506. As seen in
The inventors next tested whether FK506 could also induce S421 phosphorylation in a cell type more relevant to the disease. They therefore treated primary cultured striatal neurons with FK506 for different times and concentrations (
PolyQ Expansion Leads to Reduced Huntingtin Phosphorylation at S421 that can be Increased by FK506: Consequences on PolyQ-Induced Neuronal Toxicity
The inventors next assessed the level of phosphorylation of huntingtin in the pathological situation. They used mouse neuronal cells derived from knock-in mice in which a CAG expansion, encoding 109 glutamine residues, was inserted into the endogenous mouse huntingtin gene (109Q/109Q). This cell line closely resembles the situation in HD patients as, in these cells, polyQ-huntingtin is expressed at endogenous levels. The inventors performed immunoblotting experiments on +/+ or 109Q/109Q cells using our anti-phospho-huntingtin-S421-714 antibody and observed that phosphorylation of polyQ-huntingtin was drastically reduced compared to wild-type huntingtin (
Since phosphorylation of huntingtin is crucial to regulating polyQ-huntingtin-induced toxicity in vitro and in vivo and considering that phosphorylation is reduced in disease models, the inventors aimed to determine whether FK506 administration could induce the phosphorylation of huntingtin in vivo. Mice were treated intraperitoneally or orally with FK506 and sacrificed at different times after administration. Brains were processed for immunoblotting and analyzed for huntingtin phosphorylation at S421 (
To test the possibility that the IGF-1/Akt pathway could regulate huntingtin-mediated transport in the context of HD, the inventors analyzed the dynamics of BDNF-containing vesicles using fast 3D videomicroscopy followed by deconvolution. Primary cultures of cortical neurons were transfected with BDNF-eGFP and either wild-type full length htt (wt-FL-htt, with 17Q), polyQ-FL-htt (with 75Q) or the corresponding vector. The inventors monitored the movement of BDNF-containing vesicles by acquiring images in 3D. After deconvolution, a 2D reconstruction of each time point was performed and individual vesicles were tracked to determine the two most relevant dynamic parameters of intracellular transport. The first parameter is the mean velocity of vesicles when they are moving (i.e., with speeds superior to 0) and the second correspond to the percentage of pausing time of vesicles (time spent by the vesicles without moving). As previously reported, compared to wt-FL-htt, the presence of a polyQ expansion in htt results in a decrease in the mean velocity and an increase in the pausing time of BDNF-containing vesicles. Treatment of neurons with IGF-1 induced a statistically significant increase in the velocity of BDNF vesicles and decreased the pausing time of the vesicles (
The inventors next assessed the consequences of IGF-1 treatment in the HD genetic situation using neuronal cell lines derived from knock-in mice where a CAG expansion has been inserted into the endogenous mouse htt gene. These cell lines carry either two copies of wild-type htt (wild-type neuronal cells, +/+) or two copies of mutant htt (homozygous mutant neuronal cells, 109Q/109Q). These cell lines reflect the closest situation to HD patients as in these cells, wild-type or polyQ-htt are expressed at endogenous levels. While the velocity and pausing time of BDNF vesicles is significantly decreased in 109Q/109Q cells compared to +/+ cells (
The inventors next tested whether Akt has a beneficial effect on intracellular transport in HD pathological situation. By cotransfection experiments of Akt and BDNF-eGFP, they found that Akt completely rescues intracellular transport in 109Q/109Q cells by increasing velocity and decreasing pausing time of BDNF-containing vesicles back to wild-type values (
The inventors next investigated the mechanisms by which IGF-1 and Akt rescue the defect of BDNF transport in HD mutant cells. Previously, they have shown that Akt phosphorylates huntingtin at S421 and that phosphorylation at this residue regulates polyQ-htt-induced toxicity in neuronal cells {Humbert, 2002}. In these experiments, they found that the neuronal toxicity induced by a N-terminal 480 amino acid fragment that contains a polyQ stretch is abrogated in vitro by phosphorylation at S421. Therefore, the inventors first investigated whether such N-terminal fragment is able to promote transport and whether it is affected by polyQ expansion. They analyzed in neuronal cells the ability of 480 fragments with a normal (17Q) or expanded polyQ stretch (68Q) and compared the dynamics of intracellular transport to those obtained with wt- and polyQ-FL-htt constructs. Interestingly, they found that wild type 480 fragment of htt is able to increase the velocity and to decrease the pausing time of BDNF-containing vesicles as efficiently as the full length fragment of wild type htt (
The inventors next analyzed the consequences of the absence of phosphorylation at S421 by using constructs that contain a serine to alanine mutation. Strikingly, they found that when wild type htt (480-17) is not phosphorylated at S421, it is no longer able to promote intracellular transport as demonstrated by the decrease in velocity and the increase in the percentage of pausing time (
The alteration in the transport of BDNF in 109Q/109Q cells is rescued by Akt (
Taken together, these results indicate that the functional activity of htt on transport is regulated by its phosphorylation at S421. In addition, the inventors demonstrate that the alteration of transport in disease situation can be restored by Akt through phosphorylation of S421 of htt.
The inventors showed in the present invention that phosphorylation of huntingtin at S421 is crucial to regulating disease progression in vivo. Using a lentiviral approach to deliver N-terminal fragments of huntingtin directly to the striatum, they demonstrated that a construct encoding polyQ-huntingtin that is constitutively phosphorylated (480-68Q-S421D) induces a smaller DARPP-32-depleted region compared to a polyQ-huntingtin with an intact serine 421. Conversely, a polyQ-huntingtin that cannot be phosphorylated (480-68Q-S421A) is more toxic. The inventors have previously identified Akt and SGK as being able to act positively on S421 (Humbert et al., 2002; Rangone et al., 2004). They also reported that Akt is cleaved in post-mortem brain samples from HD patients (Humbert et al., 2002) and found that during disease progression, Akt activity is downregulated (Colin et al., 2005) further indicating that a reduced phosphorylation of huntingtin by Akt could occur in HD. In agreement, a decreased phosphorylation of polyQ-huntingtin at S421 is observed in YAC transgenic mice containing the polyQ expansion and in a cellular model of HD (Warby et al., 2005) (
The phosphorylation of a particular residue usually depends on a balance in the activities of tightly regulated kinases and phosphatases. The observed reduction in huntingtin phosphorylation could result from a reduction in Akt activity (Colin et al., 2005) but also from an excess of calcineurin phosphatase activity. Indeed, calcineurin phosphatase is highly expressed in the striatum, and in particular in the medium-size spiny neurons (MSNs), the first cells to degenerate in HD (Goto et al., 1989) and could therefore predispose huntingtin to dephosphorylation in these cells. Calcineurin is activated by Ca2+ (Mansuy, 2003) and several studies indicate that excessive Ca2+ entry in striatal neurons could play a role in HD. PolyQ-huntingtin facilitates the activity of type I inositol 1,4,5-triphosphate receptor (InsP3R1) and NR2B subtype NMDA receptor, thereby leading to increased cytosolic Ca2− levels. In MSNs the effect of mutant huntingtin on InsP3R requires Huntingtin-associated protein-1, HAP1. As the connection between disturbed Ca2− signalling and apoptosis is well established, it was proposed that glutamate released from corticostriatal projecting neurons elicits a supranormal Ca2+ response in MSNs from HD patients, leading to the opening of the mitochondria permeability transition pore (MPTP) and the activation of the caspase-dependent apoptotic cascade. In the present invention, the inventors propose that in addition to this mechanism, Ca2+, by activating calcineurin leads to dephosphorylation of huntingtin at S421 and participates in the pathogenic mechanism. In agreement they showed that an increase in cytosolic Ca2− concentration leads to dephosphorylation of huntingtin that can be blocked by FK506 demonstrating that the Ca2+-dependent dephosphorylation of S421 is dependent on CaN activity. Interestingly, another pathway by which calcineurin is activated in HD could involve calpain. Calpain is activated in HD. Calpain can regulate calcineurin activity either directly by cleaving calcineurin into an active form or by cleaving and inactivating Cabin1/cain, a potent endogenous inhibitor of calcineurin. Taken together, for the first time in HD, this study links excitotoxicity and Ca2+ to the phosphorylation of huntingtin.
The inventors' findings on the beneficial role of huntingtin phosphorylation at S421 on disease progression in vivo indicate that promoting huntingtin phosphorylation might have an impact on disease progression in HD patients as huntingtin phosphorylation is decreased when huntingtin contains the pathological polyQ expansion (Warby et al., 2005) (
In this study, the inventors focused on FK506 and found that FK506 is effective in inducing phosphorylation of huntingtin at S421 both in vitro and in vivo and in blocking polyQ-huntingtin-induced neuronal death. This suggests that FK506 is of therapeutic interest for HD patients.
As indicated previously, FK506 crosses the blood-brain barrier and, unlike CsA, does not require direct brain delivery of the compound. The fact that FK506 is routinely used in grafting procedures have established the safety and tolerability of such compounds and therefore could serve as a basis for similar therapeutic trials in HD.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06290120.2 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/050488 | 1/18/2007 | WO | 00 | 6/30/2008 |
