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WO2012117334A1 - Modulateurs allostériques positifs de mglur5 pour l'utilisation dans le traitement du syndrome de phelan-mcdermid - Google Patents

Modulateurs allostériques positifs de mglur5 pour l'utilisation dans le traitement du syndrome de phelan-mcdermid Download PDF

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WO2012117334A1
WO2012117334A1 PCT/IB2012/050894 IB2012050894W WO2012117334A1 WO 2012117334 A1 WO2012117334 A1 WO 2012117334A1 IB 2012050894 W IB2012050894 W IB 2012050894W WO 2012117334 A1 WO2012117334 A1 WO 2012117334A1
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neurons
shank3
shshank3
mglur5
dhpg
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WO2012117334A8 (fr
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Alexander DITYATEV
Elena DVORETSKOVA
Carlo Sala
Chiara VERPELLI
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Consiglio Nazionale delle Richerche CNR
Fondazione Istituto Italiano di Tecnologia
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Fondazione Istituto Italiano di Tecnologia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention refers to a novel therapeutic application for molecules having activi- ty as positive allosteric modulators of the metabotropic glutamate receptor mGlu 5.
  • Phelan-McDermid syndrome also referred to as the 22ql3 deletion syndrome, is a genetic disease without cure so far, which causes a serious form of mental retardation and autism. This syndrome is also characterized by neonatal hypotonia, overall retardation in develop- ment, seriously retarded speech ability, and minor dysmorphic features.
  • Phelan-McDermid syndrome is caused by the 22ql3 chromosome deletion.
  • the absence of one copy of the Shank3 gene (also called PROSAP2) encoding the structural protein Shank3 - which is localized in the postsynaptic density (PSD) and is in- volved in maintaining the hippocampal neuron dendritic spines - is thought to represent one of the essential causes of the main 22ql3 deletion-associated neurological features.
  • Shank is a large multi-domain protein of the postsynaptic density (PSD) scaffold, consisting of one ankyrin repeat near the N-terminus, followed by SH3, one PDZ domain, a long proline-rich region, and one SAM (sterile alpha motif) domain at the C-terminus.
  • PSD postsynaptic density
  • Shank proteins (which are coded by three genes, Shankl -3) bind to two glutamate receptors: NMD A receptors and type I metabotropic glutamate receptors (mGluRs).
  • Shank's PDZ domain binds to GKAP's C-terminus.
  • Homer interaction with the proline-rich domain ensures the association of Shank with type I metabotropic glutamate receptors (mGluRs), that is mGIuRl and mGluR5.
  • Shankl and Shank3 might form a struc- tural framework in the PSD by using different molecular mechanisms.
  • ShankS gene The connection between disruption of the ShankS gene and the neurological deficiency related to Phelan-McDermid syndrome was reported for the first time in 2001.
  • Such a con- nection is strongly supported by the finding that all of the 22ql3 deletions analyzed but one result in Shank3 deletion, either for the existence of a recurrent breakpoint within the Shank3 gene or for the recent finding that Shank3 mutations may cause a speech and/or social interaction deficiency.
  • Other small missense mutations or small interstitial deletions in Shank3 have been strongly linked to mental retardation and autistic-type disorders.
  • the present invention is based on the findings by the present inventors in connection with rat and mouse hippocampal and cortical cell cultures, whereby it has been discovered that, contrary to what is described in the state of the art (see, in particular, Roussignol et al., 2005, supra), inhibition of Shank3 expression induces a specific decrease in the expression of the metabotropic glutamate receptors mGluR5, but not of other synaptic proteins, as well as a decrease in ERKl/2 and CREB phosphorylation induced by DHPG (a type I metabotropic glutamate receptor agonist) and in mGluR5 -dependent synaptic plasticity, and also a modulation of the neural network activity.
  • DHPG a type I metabotropic glutamate receptor agonist
  • a positive allosteric modulator such as preferably CDPPB (3-cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide), results in a rescue of the synaptic functionality in Shank3 knock-down neurons.
  • a positive allosteric modulator of mGluR.5 is effective in compensating for the activity deficiency of mGluR5 receptors which is linked to Phelan-McDermid syndrome.
  • the present invention relates to a positive allosteric modulator of mGluR5 for use in the therapeutic treatment of Phelan-McDermid syndrome.
  • the positive allosteric modulator of mGluR5 is the CDPPB (3- cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide) molecule.
  • the positive allosteric modulator of mGluR5, (preferably CDPPB), is used in the therapeutic treatment of the cognitive dysfunctions of Phelan- McDermid syndrome.
  • the CDPPB molecule is preferred as it is an anti-psychotic drug that is already commercially available and, from the pharmacological point of view, it is a molecule capable of penetrating the brain.
  • ADX-47273 a molecule having activity as positive allosteric modulators of mGluR5, per se known, suitable for use in the therapeutic treatment of Phelan-McDermid syndrome according to the present invention are ADX-47273, CPPHA, VU-29, VU-36, VU-1545, DFB (l-(3-fluorophenyl)-N-((3-fluorophenyl)methylideneamino)methaneimine).
  • the positive allosteric modulator of mGluR5 may be formulated into any dosage form per se known, for instance a dosage form suitable for the already known therapeutic applications of positive allosteric modulators of mGluR5, particu- larly CDPPB.
  • a person of ordinary skill in the art is able to modify the characteristics of the dosage form according to need, without requiring undue experimentation or the exercise of any inventive skill.
  • the determination of the amount of active principle to be administered to the patient which depends on several factors related to both the disease and the characteristics of the patient, also is within the skills of the person of ordinary skill in the art (as above).
  • a therapeutically effective dose of CDPPB may be comprised between 0.1 and 100 mg/kg of body weight for a human patient suffering from Phelan-McDermid syndrome.
  • An effective concentration of CDPPB in the experiments performed in vitro is comprised between about 0.1 and 200 ⁇ .
  • RNA interference RNA interference
  • the inven- tors observed a decrease in mGluR5 signaling (decrease in ERK1/2 and CREB phosphorylation induced by stimulation with DHPG as a mGluR5 agonist), a reduction in mGluR5- dependent synaptic plasticity, and a decrease in mGluR5 -dependent modulation of the neural network activity.
  • the inventors also found morphological abnormalities in the synaptic structure (number, length and width of the spines) and a decrease in the glutamatergic syn- aptic transmission (reduction of the frequency of the miniature excitatory postsynaptic currents, mEPSC).
  • the pharmacological increase in mGluR5 activity through use of CDPPB (3-cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide) as a positive allosteric modulator of these receptors, restores mGluR5 -dependent signaling (ERK1/2 phosphorylation induced by DHPG) and normalizes the frequency of the miniature excitatory postsynaptic currents in Shank3 knock-down neurons.
  • CDPPB 3-cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide
  • FIG. 1 The decrease (knock-down) in Shank3 expression impairs the expression of mGluR5.
  • A Hippocampal neurons on DIV 7 were infected with a lentivirus expressing shShank3 or with shCtrl, as indicated above the panels. After one week, the neurons were solubilized and analyzed by Western blotting with the antibodies indicated on the left-hand side of the panels.
  • B Average levels ( ⁇ SEM) of the proteins (normalized against non- infected neurons) in the hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl; at least four independent experiments were performed.
  • the top histogram relates to the results obtained with reference to the non-infected control
  • the middle histogram relates to the re- suits obtained with reference to infection with shCtrl
  • the bottom histogram relates to the results obtained with reference to infection with shShank3.
  • the expression levels of Shank3 and mGluR5 were significantly lower in shShan3 -infected neurons compared to non-infected and shCtrl-infected neurons; *p ⁇ 0.01, Student's t-test. At least four independent experiments were performed.
  • (C) Synaptosomes were obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl and were analyzed by Western blotting with the antibodies indicated on the left-hand side of the panels.
  • the top histogram relates to the results obtained with reference to the shCtrl infection, and the bottom histogram relates to the re- suits obtained with reference to infection with shShank3.
  • the expression levels of Shank3 and mGluR5 were significantly lower in shShan3 -infected neurons compared to shCtrl- infected neurons; *p ⁇ 0.01, Student's t-test.
  • FIG. 1 The decrease (knock-down) in Shank3 expression modifies mGluR5 signaling.
  • A Hippocampal neurons on DIV 7 were infected with a lentivirus expressing shShank3 or shCtrl. After one week, the neurons were treated with DHPG 100 ⁇ , NMDA 100 ⁇ or KCl 50 mM for 30 minutes, as indicated above the panels, and thereafter solubilized and analyzed by Western blotting for Shank3, pERKl/2, ERK1/2, pCREB and CREB expression, as indicated on the left-hand side of the panels.
  • the panels show confocal microscopy images of hippocampal neurons transfected on DIV 7 with shCtrl, shShank3 or shShank3 plus shShank3r (shShank3 -resistant form), as indicated on the left-hand side of the panels, and treated on DIV 14 with DHPG 100 ⁇ or KCl 50 mM, as indicated above the panels.
  • the neurons were fixed and stained for GFP and pERKl/2.
  • D In each of the transfection and treatment conditions, pERKl/2 signals were quantified as described in Materials and Methods; the average values are shown as bars ( ⁇ SEM).
  • the left histogram relates to the results obtained with reference to the shCtrl infection
  • the central histogram relates to the results obtained with reference to the shShank3 infection
  • the right histogram relates to the results obtained with reference to the shShank3 + Shank3r infection.
  • Shank3 expression decreases the frequency but not the amplitude or time course of mEPSCs in cultured hippocampal neurons.
  • Aa Representative mEPSCs recorded on DIV 7 from control neurons (shCtrl, left) and after Shank3 knock down (shShank3, right) at a starting potential of -60 mV in the presence of TTX 0.5 ⁇ and picrotoxin 50 ⁇ .
  • TTX 0.5 ⁇
  • picrotoxin 50 ⁇ Some selected time intervals are shown with a higher time resolution.
  • Ac Averaged mEPSCs obtained from a control neuron (left) and from a shShank3 -treated neuron (right).
  • FIG. 4 Reduction in the long-term depression (LTD) induced by DHPG and decrease in the GluRl subunit expression of AMPA receptors in hippocampal shShank3 -treated neu- rons.
  • LTD long-term depression
  • A Examples of mEPSCs recorded immediately before (left) and 30 minutes after (right) the beginning of DHPG application. The mEPSCs were recorded at a starting potential of -60 mV in the presence of TTX 0.5 ⁇ and picrotoxin 50 ⁇ .
  • aminophosphovaleric acid (APV) 50 ⁇ was applied together with DHPG 100 ⁇ or applied alone as a control.
  • mEPSC frequencies of shCtrl-treated neurons and shShank3 -treated neurons are significant (*p ⁇ 0.05, Student's t-test).
  • D mEPSC amplitudes were not affected by DHPG in shCtrl-treated neurons or in shShank3 -treated neurons.
  • E Examples of GluRl immunostaining on the cell surface in hippocampal neurons on DIV 14. The neurons were treated with DHPG 100 ⁇ (+DHPG) for 10 minutes, or were not treated (-DHPG). Thereafter, the viable neurons were stained with anti-GluRl antibodies for 15 minutes (E, top). A decrease in GluRl expression on the cell surface of control neurons (shCtrl) is seen after treatment with DHPG.
  • Shank3-specific shRNA Characterization of Shank3-specific shRNA.
  • A Hippocampal neurons on DIV 7 were infected or not with a Shank3 shRNA-expressing lentivirus (shShank3); after one week, the neurons were analyzed by Western blotting with antibodies (as indicated on the left-hand side of the panels) specific for Shankl, Shank2 and Shank3, or with antibodies that recognize all three Shank proteins (PanShank).
  • B Average levels ( ⁇ SEM) of Shank protein (normalized against levels in non-infected neurons) in hippocampal neurons infect- ed or not with a lentivirus expressing shShank3; at least four independent experiments were performed.
  • Shank3 and PanShank levels were significantly lower in shShank3- infected neurons compared to non-infected neurons, *p ⁇ 0.01, Student's t-test.
  • C Average levels ( ⁇ SEM) of Shank mRNA (normalized against levels in non-infected neurons) in hippocampal neurons infected or not with a lentivirus expressing shShank3 or control shRNA (shCtrl); at least four independent experiments were performed.
  • Shank3 mRNA level was significantly lower in shShank3 -infected neurons compared to non-infected or shCtrl-infected neurons, *p ⁇ 0.01, Student's t-test.
  • D Hippocampal neurons on DIV 7 were infected or not with a shShank3- or shCtrl-expressing lentivirus and stained after one week with antibodies (as indicated on the right-hand side of the panels) specific for Shank3, synaptophysin, and PSD-95 clusters in hippocampal neurons infected with a shShank3- or shCtrl-expressing lentivirus; at least four independent experiments were performed and at least five neurons per experiment were considered.
  • the left histogram relates to the results obtained with reference to the non-infected control
  • the central histogram relates to the results obtained with reference to infection with shCtrl
  • the right histogram relates to the results obtained with reference to infection with shShank3.
  • the number of Shank3 clusters was significantly lower in shShank3 -infected neurons compared to non-infected or shCtrl-infected neurons, *p ⁇ 0.01, Student's t-test.
  • FIG. 7 COS cells were transfected with HA-Shank3, GFP-Shank3r, GFP- Shank3R87Cr or GFP-Shank3InsGr with or without shShank3, as indicated above the pan- els, then solubilized and analyzed by Western blotting with anti-HA, anti-GFP or anti- tubulin antibodies.
  • B Average levels ( ⁇ SEM) of PSD-95, GluRl and GluR2 mRNAs (normalized against levels in non-infected neurons) in hippocampal neurons infected or not with a lentivirus expressing shShank3 or control shRNA (shCtrl); at least four independent experiments were performed.
  • the left histogram relates to the results obtained with reference to the non- infected control
  • the central histogram relates to the results obtained with reference to in- fection with shCtrl
  • the right histogram relates to the results obtained with reference to infection with shShank3.
  • FIG. 8 Morphology of the dendritic spines in hippocampal neurons infected with shShank3.
  • A Hippocampal neurons on DIV 7 were transfected with shCtrl and several cDNAs expressing DsRed, as indicated on the right-hand side of the panels. After one week, the neurons were fixed and stained for GFP and DsRed.
  • B Quantification ( ⁇ SEM) of the dendritic spine numbers (per 10 ⁇ ), length and width. Over 14 transfected neurons from four independent experiments were measured for each transfection.
  • FIG. 9 Shank3 mutations observed in autistic patients do not restore siShank3 -induced deficiencies in mGluR5 pathway.
  • the panels show confocal images of hippocampal neurons transfected on DIV 7 with siShank3+Shank3R87Cr or siShank3+Shank3InsGr, as indicated on the left-hand side of the panels; the cells were fixed and stained for GFP and pERKl/2.
  • B The signals obtained for pERKl/2 in each of the transfection and treatment conditions were quantified as described in Materials and Methods; the average values are shown as bars ( ⁇ SEM).
  • the left histo- gram relates to values obtained from shCtrl infection
  • the central histogram (left) relates to values obtained from shShank3 infection
  • the central histogram (right) corresponds to values obtained from infection with shShank3+Shank3R87Cr
  • the right histogram relates to values obtained from infection with shShank3+Shank3InsGr.
  • FIG. 11 The decrease (knock-down) in Shank3 expression impairs DHPG-induced modulation of the cortical network activity.
  • A Representative 60-electrode recordings of activity of cultured mouse cortical neurons before and 5 minutes after application of 100 ⁇ DHPG. Each horizontal line corresponds to one electrode; short vertical intervals correspond to detected action potentials. The duration of each recording is 30 seconds. Following application of 100 ⁇ DHPG, a greater increase in the number of bursts occurs in the shCtrl-treated cultures compared with the shShank3 -treated cultures.
  • B-E Changes in the mean firing rate (B), mean bursting rate (C), intra-burst firing rate (D), and percentage of out-burst spikes (E) for active electrodes after the application of DHPG to shCtrl- and shShank3 -treated cultures.
  • the curve marked by an "a” relates to shCtrl and the one marked by a "b” relates to shShank3.
  • Hippocampal neuronal cultures and chemical reagents for the biochemical experiments Hippocampal neuronal cultures were prepared from 18- or 19-day-old rat embryos obtained from Charles River Laboratories.
  • High density (750-1000 cells/mm 2 ) and medium density (150-200 cells/mm 2 ) neurons were plated and grown as described in Romorini et al., JNeurosci 2004; 24(42):9391-9404, using B27 prepared in the laboratory.
  • the neurons were plated onto 6-well tissue culture plates (Iwaki, Bibby Sterilin), or 18- mm coverslips and grown on 12-well tissue culture plates (Iwaki, Bibby Sterilin).
  • the cultures were infected with lentivirus expressing shRNA specific for luciferase (shCtrl) or Shank3 (shShank3) after 7 days in vitro ("days in vitro" hereinafter abbreviated as "DIV") or transfected using a calcium phosphate precipitation protocol according to the protocol described in Sala C et al, Neuron 2001; 31(1):1 15-130.
  • the cells were treated with 100 ⁇ DHPG (group I metabotropic glutamate receptor agonist), 100 ⁇ NMD A, or 50 mM KC1 at 15 DIV for 30 minutes.
  • 2 ⁇ tetrodotoxin was added to the cultures 12 hours before stimulation.
  • neurons were treated for 12 hours with 100 nM or 1 ⁇ CDPPB before DHPG stimulation.
  • Neurons or COS-7 cells were solubilized in Laemmli buffer and loaded onto a 6-12% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes (BioRad) at 80 V for 120 minutes at 4°C. Primary antibodies were applied overnight in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20 and 3% dried skimmed milk or BSA). Secondary antibodies (HRP-conjugated anti-mouse, anti-rabbit or anti-goat or anti-guinea pig, GE Healthcare) were used at a 1 :2000 dilution. The signal was detected using an ECL detection system (PerkinElmer Life Sciences).
  • the total intensity of the bands was measured with ImageJ software.
  • the signal intensities of the proteins were normalized according to a signal from actin or tubulin; the intensity of the phosphospecific ERK1/2 immunoreactivity was normalized against the total ERKl/2 signal in the same run. Changes in protein levels and in ERK1/2 phosphorylation were compared with those of untreated samples and were expressed as fold-increase or decrease. The results are shown as mean ⁇ SEM.
  • rabbit anti-Shank3 1 :1000 (Santa Cruz Biotechnology); guinea pig anti-Shank3; rabbit anti-ERKl/2 1 :500, rabbit anti-pERK 1/2 1 :500, rabbit anti-eEF2 1 :1000, and rabbit anti-GFP 1 :500 (Cell Signaling Technology); rabbit anti-mGluR5 1 :600 and rabbit anti-GluR2/3 1 :250 (Millipore Bioscience Research Reagents); rabbit anti-GluR2 1 :400, mouse anti-Shank 1 1 :600, mouse anti-Shank2 1 :600, mouse anti-Pan Shank 1 :600, mouse anti-PSD95 1 : 10000 (NeuroMab, * UC Davis/NIH NeuroMab Facility); rabbit anti-GKAP 1 :500 (gift from Morgan Sheng, Genentech); mouse anti-synaptophysin 1 :1000, mouse anti-P-
  • neurons were fixed in 4% paraformaldehyde and 4% sucrose at room temperature or in 100% methanol at -20 °C.
  • Primary and secondary antibodies were applied in GDB buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl) for 2 hours at room temperature or overnight at 4 °C.
  • rabbit anti-pERK 1 100 (Cell Signaling Technology); mouse anti-Pan Shank 1 :200 (NeuroMab, UC Davis/NIH NeuroMab Facility); guinea pig anti-Shank3; mouse anti-GFP 1 :500 (Roche); and secondary antibodies conjugated to FITC, Cy3 and Cy5 (Jackson ImmunoResearch).
  • Shank3 oligonucleotides were annealed and inserted into the Hindlll/Bglll sites of the pLVTHM vector for lentivirus production.
  • siRNA sequences that target rat Shank3 mRNA were used (GenBank accession number NM_021676): 5 ' GGAAGTC ACC AGAGGAC AAGA3 ' .
  • the Shank3 rescue (Shank3r), R87C (Shank3R87Cr), and InsG (Shank3InsGr) constructs resistant to interference by siRNA were generated by changing six nucleotides in the siRNA target site, without changing the amino acid sequence of the protein.
  • Shank3 R87C and InsG mutants have been described in Durand CM et al., Nat Genet 2007; 39(1): 25-27. Measurement of dendritic spine morphology and ERK1/2 phosphorylation
  • Neurons were cotransfected with an siRNA vector and DsRed at a ratio of 2: 1 (7.5 ⁇ g of total DN A/well in 12- well plates) on DIV 7 and fixed on DIV 18. A few labeled transfected neurons were randomly chosen for quantification in at least four independent experiments for each construct.
  • the cells were then plated at a density of 300/mm 2 in neurobasal-A medium supplemented with 5 ⁇ g/ml gentamicin, 2% B27 supplement, 25 ⁇ g/ml FGF2, and 0.5 mM L-glutamine (all from Invitrogen) onto 18 mm-diameter round glass coverslips (Menzel-Glaser) coated overnight with 100 ⁇ g/ml poly-L-lysine (Sigma- Aldrich) and 40 ⁇ g ml laminin (Sigma- Aldrich).
  • the cultures were maintained at 37°C in a humidified incubator with 95% 0 2 and 5% C0 2 . Beginning from day 3 in culture, the medium was supplemented with 0.5 ⁇ AraC (Sigma-Aldrich) to prevent glial cell proliferation. The medium was changed twice a week. Infection and transfection of neurons
  • the neuronal cultures were incubated with the DNA-calcium phosphate precipitate for about 1.5 hours. After incubation, the precipitate was dissolved by incubation for 15 minutes in a medium that had previously been equilibrated in an incubator with 10% C0 2 . The plates were then returned to their original conditioned medium and the following day the expression of the tdTomato protein was checked. The cells were used for electrophysiological recordings 3-4 days after transfection.
  • the allosteric modulator of mGluR5 (CDPPB, 1 ⁇ , dissolved in 0.1% DMSO) was applied overnight on DIV 13 and during recordings of mEPSCs on DIV 14. As a vehicle control, 0.1% DMSO was used.
  • Electrodes with a resistance in the range of 3-6 MOhms were filled with a solution that contained 130 mM CsMeS0 4 , 8 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 0.5 mM EGTA, and 10 mM HEPES, pH 7.25.
  • HBS HEPES -buffered saline
  • mM 1 19 NaCl, 5 KC1, 2 CaCl 2 , 2 MgCl 2 , 25 HEPES, 33 D-glucose, 0.0005 tetrodotoxin citrate (Tocris), and 0.05 picrotoxin (Tocris), pH 7.35.
  • the osmolarity of HBS was adjusted to that of the culture medium on the day of recording.
  • the osmolarity of the electrode solution was 10 mOsm minus that of HBS.
  • DHPG 100 ⁇ , Tocris
  • the detection threshold for GluRl -positive fluorescent clusters was fixed at twice the level of background fluorescence obtained from a region of diffuse fluorescence within the dendritic shaft. Only clusters lying along secondary dendritic branches were counted; regions in which the identification of neuronal processes was ambiguous were excluded from the quantification. For quantification, the number of GluRl immunoreactive clusters per 100- ⁇ dendritic length within a given field was used. Measurements obtained from 15-25 dendrites, corresponding to a certain condition (i.e. shCtrl or shShank3, before or after DHPG), from each culture preparation were averaged and the values from four independent preparations were used for a statistical comparison between the groups, performed by two-way ANOVA analysis and t-test for paired data.
  • Microelectrode Array Recordings and Analysis Microelectrode arrays (Multichannel Systems, MCS, Reutlingen, Germany) consisted of 60 TiN/SiN planar round electrodes (30 ⁇ diameter, 200- ⁇ center to center interelectrode distance) arranged in an 8 ⁇ 8 square grid excluding corners. Dissociated cortical neurons from PI C57BL6/J mice were prepared and plated. The cultures were infected on DIV 8-10, as described above. The activity of all cultures was recorded using the MEA60 System (MCS). After l200 amplification, signals were sampled at 10 kHz and acquired through the data acquisition card and MC_Rack software (MCS).
  • MCS MEA60 System
  • MEAs were kept at 37 °C by means of a controlled thermostat (MCS) and covered by flexible polydimethylsiloxane lids, to avoid evaporation and prevent changes in osmolality.
  • MCS controlled thermostat
  • One recording session per culture was done. The session included 30 minutes of baseline recordings in the absence of DHPG and three consecutive 30-minute recordings in the presence of 1 ⁇ , 10 ⁇ e 100 ⁇ DHPG. The last 20 minutes of each episode were analyzed to exclude the initial part of the recordings, during which neuronal activity may have been influenced by the mechanical disturbances evoked by injection of the drug. Only cultures in which more than 70% Shank3 expression was knocked out were included in the analysis.
  • the spike trains were analyzed using a custom burst detection method (Pasquale V et al., J Comput Neurosci 2010; 29(1-2):213-219), the parameters of which are directly estimated from the inter-spike interval distribution of each channel.
  • burst detection procedure several measures describing spike and burst statistics were extracted; these included mean firing rate, mean bursting rate, mean burst duration, mean frequency intra- burst (spikes/second), and percentage and frequency of out-burst spikes, i.e. spikes not included in bursts over the total.
  • Two-way ANOVA analysis with repeated measures followed by Holm-Sidak pairwise comparison test of groups was used for statistical evaluation of the DHPG and shShank3 effects.
  • Shank3 expression was knocked down using RNA interference.
  • Shank3 shRNA (shShank3) strongly reduced the levels of endogenous Shank3 mRNA and protein, but not those of other Shank family members in hippocampal cultures compared to a control shRNA (shCtrl) ( Figures 6A-C).
  • Immunocytochemical staining with Pan-Shank antibody showed approximately a 40% decrease in total Shank immunoreactivity in total ly- sates ( Figures 6A-B) obtained from shShank3 -infected neurons.
  • Shank3 -specific bands have a lower molecular weight than that of the higher band at 240 kD, which probably is Shank 1.
  • the numbers of synaptophysin and PSD-95 clusters were not modified by shShank3 treatment ( Figures 1D-E).
  • a rescue experiment with an shRNA- resistant Shank3 (Shank3r) confirmed the specificity of shShank3 ( Figures 2, 7A and 10).
  • Shank3 plays an important role in assembling the PSD and in forming excitatory synapses via its multiple protein-protein interactions. Accordingly, the present inventors examined the effect of Shank3 expression knockdown on the protein composition of .excitatory synapses using synaptosome total lysates obtained from hippocampal cultures infected with shShank3 or shCtrl. By immunoblotting with a Shank3 -specific antibody, the inventors confirmed a strong reduction in Shank3 protein in the total lysate and in the synaptosomal fraction of shShank3 -infected neurons ( Figures 1 A, C). The expression of several glutamate receptors, scaffold proteins, and signaling molecules was measured by immunoblotting (Figure IB).
  • mGluR5 Activation of mGluR5 could lead to a postsynaptic LTD that is mediated by reduced synaptic expression of AMPA receptors.
  • shShank3- and shCtrl-treated cultures with 100 ⁇ DHPG.
  • DHPG induced LTD long term depression
  • mEPSC long term depression
  • knock-down of Shank3 expression impaired the long term depression (LTD).
  • shShank3 treatment impaired DHPG-induced down-regulation in the number of GluRl- immunoreactive clusters on the plasma membranes of dendrites ( Figures 4E-F); a similar down-regulation has been reported for uninfected cultured neurons.
  • the density of GluRl-immunoreactive clusters under basal conditions i.e. in neurons not treated with DHPG, was lower in shShank3 -treated cultures than in controls. This finding corroborates the observed reduction in mEPSC frequencies in shShank3 -treated cultures under basal conditions.
  • Shank3 is a large multidomain protein of the postsynaptic density scaffold, which belongs to a protein family encoded by three genes, SHANK 1, SHANK2, and SHANK3. Although the proteins encoded by these three genes are structurally similar, some evidence suggests that they differ in function, both in synapse- targeting properties and in binding partners.
  • Shankl induces maturation of dendritic spines without increasing their numbers
  • Shank3 induces the formation of new synapses and dendritic spines.
  • Shankl targeting to synapses is dependent on the PDZ domain
  • targeting of Shank2 and Shank3 depends on their C-terminal domain, including the SAM domain.
  • Shank2 and Shank3 multimerize to form a framework in the postsynaptic density (PSD) which depends on Zn 2+ ion binding to the SAM domain.
  • PSD postsynaptic density
  • Shankl does not bind Zn 2+ ions, but forms a large structural complex with Homer in the postsynaptic density (PSD).
  • Shank proteins in dendritic spines are probably related to the fact that these proteins bind directly or indirectly, through binding to Homer, to a number of proteins involved in actin remodeling, such as cortactin, Abpl, IRsP53, and SPIN90, oligophrenin, and CdC42.
  • actin remodeling such as cortactin, Abpl, IRsP53, and SPIN90, oligophrenin, and CdC42.
  • Available data suggest that Shank proteins functionally link glutamate receptors to the cytoskeleton, thereby regulating the size and form of excitatory synapses and dendritic spines.
  • Shank2 and Shank3 can also bind to Abl and LAPSER1 , two proteins that translocate from the postsynaptic density to the nucleus in an activity- dependent manner and induce gene transcription and translation.
  • the present inventors found that knockdown of Shank3 expression specifically impaired mGluR5 signaling at the synapses. In neurons knocked down for Shank3, the amount of mGluR5 protein - but not of its mRNA - is specifically reduced in the total lysate and in the synaptosomes, suggesting that Shank3 is somehow involved in mGluR5 protein stabilization.
  • Previous work has shown that mGluR5 binds to Shank3 directly, or indirectly through Homer. However, because the present inventors found no changes in Homer expression, it is possible that the direct binding of Shank3 to mGluR5 is involved in this phenomenon.
  • Shank3 and mGluR5 can be degraded by proteasomes following ubiquitination, suggesting that their interaction can reciprocally modulate their ubiquitination and stabilization.
  • the inventors did not find any change in Shank3 protein expression in knock-out mice in which mGluR5 expression had been deleted.
  • Shank3 might act as a stabilization platform for mGluR5.
  • Densin-180 binds to Shank3 and could link Shank3 to mGluR5, stabilizing the complex at the synapses.
  • the inventors also observed a reduction in cell surface expression of GluRl in shShank3- treated neurons without a reduction in its protein expression.
  • the reduction in GluRl cell surface expression correlates with the reduced mEPSC frequencies.
  • the impaired DHPG- dependent LTD observed in shShank3 -treated neurons correlates with absence of changes in cell surface expression of GluRl , which is down-regulated by DHPG in the control.
  • CDPPB an allosteric mGluR5 agonist
  • was able to rescue mEPSC frequencies in neurons knocked down for Shank3 expression these data suggest that Shank3 regulates AMP A receptor trafficking in an mGluR5 -dependent manner.
  • the reduction in cell surface GluRl expression and in mEPSC frequencies after knockdown of Shank3 might reflect impairment in activity-dependent synaptic recruitment of AMPA receptors at basal conditions.
  • Shank3 deletion at synapses specifically compromises mGluR5 signaling. Furthermore, the expression of mutated forms of Shank3 that mimic the mutations found in autistic patients was not able to rescue DHPG-dependent ERK1/2 phosphorylation. Thus, reduction in Shank3 expression (which occurs in 22ql3/Phelan-McDermid syndrome) and mutations in Shank3 (which occur in some autistic patients), might both induce alterations in mGluR5 signaling at synapses.
  • the mGluR5 receptor was found to play a major role in synaptic plasticity. It has been clearly demonstrated that antagonism or genetic deletion of mGluR5 impair both acquisition and extinction of hippocampal-dependent learning tasks, such as the radial arm maze and the Morris water maze, by impairing both the late phase of hippocampal long term potentiation and mGluR-dependent long-term depression. The occurrence of a mGluR-dependent long-term depression in CA1 relies on both ERK and PI3K-mTOR pathways. A role for mGluR-LTD has been demonstrated for the formation of object recognition memory.
  • This enhancement occurs because, in the absence of FMRP, as in fragile X syndrome, there is a loss of steady-state translational suppression, which leads to increased target mRNAs for FMRP and a resulting increase in expression levels of proteins, such as the activity-regulated cytoskeleton-associated protein (ARC) that may enhance the magnitude of LTD.
  • ARC activity-regulated cytoskeleton-associated protein
  • mGluR5 antagonists or genetic reduction of mGluR5 can reverse multiple phenotypes in mice deficient in FMR1, a gene encoding for FMRP.
  • FMR1 a gene encoding for FMRP.
  • phenotypes include increased dendritic spine density and deficiencies in experience-dependent and learning-dependent plasticity in the hippocampus and visual cortex.

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Abstract

L'invention concerne une nouvelle application thérapeutique pour des modulateurs allostériques positifs du récepteur métabotropique du glutamate mGluR5, notamment pour le traitement thérapeutique du syndrome de Phelan-McDermid provoqué par une délétion de 22qI3. Dans ce but, un modulateur allostérique positif préféré est le 3-cyano-N-(1,3-diphényl-1-H-pyrazol-5-yl)benzamide, dont l'abréviation est CDPPB.
PCT/IB2012/050894 2011-02-28 2012-02-27 Modulateurs allostériques positifs de mglur5 pour l'utilisation dans le traitement du syndrome de phelan-mcdermid Ceased WO2012117334A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014210538A1 (fr) * 2013-06-28 2014-12-31 The Regents Of The University Of California Traitement des déficits cognitifs associés au syndrome de noonan
WO2019082125A1 (fr) * 2017-10-27 2019-05-02 Amo Pharma Ltd. Méthodes de traitement du syndrome de phelan mcdermid à l'aide de farnésyl-dibenzodiazépinones

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113222975B (zh) * 2021-05-31 2023-04-07 湖北工业大学 一种基于改进U-net的高精度视网膜血管分割方法

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
AYALA J E ET AL: "MGluR5 Positive Allosteric Modulators Facilitate both Hippocampal LTP and LTD and Enhance Spatial Learning", NEUROPSYCHOPHARMACOLOGY, vol. 34, no. 9, 1 August 2009 (2009-08-01), pages 2057 - 2071, XP002640853, DOI: 10.1038/NPP.2009.30 *
BOLOGNA LL ET AL., NEUROSCIENCE, vol. 165, no. 3, 2010, pages 692 - 704
DITYATEV, A. ET AL., NEURON, vol. 26, 2000, pages 207 - 217
DURAND CM ET AL., NAT GENET, vol. 39, no. 1, 2007, pages 25 - 27
JIANG M ET AL., NAT PROTOC, vol. 1, no. 2, 2006, pages 695 - 700
MACCIONE A ET AL., J NEUROSCI METHODS, vol. 177, no. 1, 2009, pages 241 - 249
MOULT PR ET AL., JNEUROSCI, vol. 26, no. 9, 2006, pages 2544 - 2554
PASQUALE V ET AL., J COMPUT NEUROSCI, vol. 29, no. 1-2, 2010, pages 213 - 219
REICHEL CARMELA M ET AL: "Loss of Object Recognition Memory Produced by Extended Access to Methamphetamine Self-Administration is Reversed by Positive Allosteric Modulation of Metabotropic Glutamate Receptor 5", NEUROPSYCHOPHARMACOLOGY, vol. 36, no. 4, 8 December 2010 (2010-12-08), pages 782 - 792, XP002640850 *
ROMORINI ET AL., JNEUROSCI, vol. 24, no. 42, 2004, pages 9391 - 9404
ROSENBROCK HOLGER ET AL: "Functional interaction of metabotropic glutamate receptor 5 and NMDA-receptor by a metabotropic glutamate receptor 5 positive allosteric modulator", EUROPEAN JOURNAL OF PHARMACOLOGY,, vol. 639, no. 1-3, 1 August 2010 (2010-08-01), pages 40 - 46, XP002640852 *
ROUSSIGNOL ET AL., THE JOURNAL OF NEUROSCIENCE, vol. 25, no. 14, 6 April 2005 (2005-04-06), pages 3560 - 3570
ROUSSIGNOL GAUTIER ET AL: "Shank expression is sufficient to induce functional dendritic spine synapses in aspiny neurons", JOURNAL OF NEUROSCIENCE, vol. 25, no. 14, 6 April 2005 (2005-04-06), pages 3560 - 3570, XP002640849 *
SALA C ET AL., NEURON, vol. 31, no. 1, 2001, pages 115 - 130
VALES KAREL ET AL: "The difference in effect of mGlu2/3 and mGlu5 receptor agonists on cognitive impairment induced by MK-801", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 639, no. 1-3, 1 August 2010 (2010-08-01), pages 91 - 8, XP002640851 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014210538A1 (fr) * 2013-06-28 2014-12-31 The Regents Of The University Of California Traitement des déficits cognitifs associés au syndrome de noonan
US10835513B2 (en) 2013-06-28 2020-11-17 The Regents Of The University Of California Methods and treatments for the learning and memory deficits associated with Noonan syndrome
WO2019082125A1 (fr) * 2017-10-27 2019-05-02 Amo Pharma Ltd. Méthodes de traitement du syndrome de phelan mcdermid à l'aide de farnésyl-dibenzodiazépinones
CN111601602A (zh) * 2017-10-27 2020-08-28 阿默制药有限公司 使用法呢基二苯并二氮杂酮治疗Phelan McDermid综合征的方法
AU2018357347B2 (en) * 2017-10-27 2024-02-29 Amo Pharma Ltd. Methods of treating Phelan McDermid Syndrome using farnesyl dibenzodiazepinones

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