WO2012117334A1

WO2012117334A1 - Mglur5 positive allosteric modulators for use in the treatment phelan-mcdermid syndrome

Info

Publication number: WO2012117334A1
Application number: PCT/IB2012/050894
Authority: WO
Inventors: Alexander DITYATEV; Elena DVORETSKOVA; Carlo Sala; Chiara VERPELLI
Original assignee: Consiglio Nazionale delle Richerche CNR; Fondazione Istituto Italiano di Tecnologia
Current assignee: Consiglio Nazionale delle Richerche CNR; Fondazione Istituto Italiano di Tecnologia
Priority date: 2011-02-28
Filing date: 2012-02-27
Publication date: 2012-09-07
Anticipated expiration: 2013-08-28
Also published as: ITTO20110169A1; WO2012117334A8; IT1404270B1

Abstract

The invention concerns a novel therapeutic application for positive allosteric modulators of the metabotropic glutamate receptor mGluR5, notably for the therapeutic treatment of Phe- lan-McDermid syndrome caused by 22qI3 deletion. For this purpose, a preferred positive allosteric modulator is 3-cyano-N-(1,3-diphenyl-1-H-pyrazol-5-yl)benzamide, abbreviated as CDPPB.

Description

MGLUR5 POSITIVE ALLOSTERIC MODULATORS FOR USE IN THE TREATMENT OF PHELAN-MCDERMID SYNDROME

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.

As indicated above, 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.

At the molecular level, 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.

In these last years, the role of Shank proteins has been studied by many researchers, which reported that over-expression of Shankl in hippocampal neurons increases the maturation rate of filopodium-like spikes into mature spines and promotes the enlargement of the mature spines. In contrast, mice lacking Shankl display smaller dendritic spines, weaker synaptic transmission, and altered spatial learning. Shankl and Shank3 might form a struc- tural framework in the PSD by using different molecular mechanisms.

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. More recently, other small missense mutations or small interstitial deletions in Shank3 have been strongly linked to mental retardation and autistic-type disorders.

Roussignol et al., The Journal ofNeuroscience, April 6, 2005, 25(14):3560-3570, showed that over-expression of Shank3 in cerebellum granules induced formation of dendritic spines and synapses by recruitment of glutamate receptors, whereas inhibition of Shank3 expression in hippocampal neurons decreased the number of dendritic spines. However, this publication does not indicate or suggest that a decrease in the Shank3 protein may reduce the specific metabotropic glutamate receptor mGluR5 in synapses. Moreover, this publication suggests that over-expression of Shank3 increases different glutamate receptor subtypes, generally suggesting that Shank3 regulates the synaptic localization of all the glutamate receptors.

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.

The studies carried out by the present inventors also showed that the pharmacological in- crease in mGluR5 activity through 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. Based on these findings, 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.

Thus, the present invention relates to a positive allosteric modulator of mGluR5 for use in the therapeutic treatment of Phelan-McDermid syndrome. In a preferred embodiment, the positive allosteric modulator of mGluR5 is the CDPPB (3- cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide) molecule.

In a further preferred embodiment, 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.

Other molecules 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).

For use as a medicament in the therapeutic treatment of Phelan-McDermid syndrome according to the present invention, 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. Anyway, 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). As a general, but not limiting, indication, 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 μΜ.

The experimental section that follows is provided solely by way of illustration and not of limitation of the scope of the present invention as defined in the appended claims, the subject of which also forms an integral part of the patent specification.

The experimental section actually describes the experiments performed and the data obtained by the present inventors whereon the novel therapeutic application, which forms the subject of the present invention, is based. In summary, the inventors employed the RNA interference (RNAi) technique to decrease (knock down) Shank3 expression in hippocampal and cortical cell cultures and showed that this treatment specifically reduces the synaptic expression of the metabotropic glutamate receptor mGluR5, but does not affect the expression of other important synaptic proteins. As a functional consequence of the knock down of Shank3 expression by RNAi, 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). In particular, 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. The results of the experiments carried out by the inventors, summarized concisely above but described in further detail in the experimental section that follows, demonstrate that a deficiency in mGluR.5 -mediated intracellular signaling in Shank3 knock-down neurons may be compensated for by a treatment with CDPPB, and therefore these form the experimental basis that supports the therapeutic application that forms the subject of the present invention.

In the following experimental section, the results of the performed experiments are described with reference to the appended drawings, the technical contents of which are briefly described hereinafter.

Figure 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. For each protein indicated on the left-hand side of the panel (B), 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, and 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. (D) Average levels (± SEM) of the proteins (normalized against shCtrl-infected neurons) in synaptosomes obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl; at least three independent experiments were performed. For each protein indicated on the left-hand side of the panel (D), 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.

Figure 2. 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 μΜ, NMDA 100 μΜ 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. (B) Average levels (± SEM) of pERKl/2 and pCREB (normalized against total ERK and CREB and against non-treated controls, NT, values); at least five independent experiments were performed. pERKl/2 and pCREB levels were significantly lower in shShank3 -infected neurons compared to shCtrl - infected neurons after treatment with DHPG, *p<0.01, Student's t-test. (C) 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 μΜ 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). For each treatment condition (NT, DHPG and KCl), 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, and the right histogram relates to the results obtained with reference to the shShank3 + Shank3r infection. pERKl/2 level after treatment with DHPG was significantly lower in shShank3 -transfected neurons com- pared to neurons transfected with shCtrl and with shShank3 plus shShank3r, *p<0.05, Student's t-test. Scale bar = 20 μιη. Figure 3. The decrease (knock-down) in 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 μΜ and picrotoxin 50 μΜ. (Ab) 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). (B, C, D, E) Summarizing graphs of mEPSC parameters in the controls («=16 cells) and in shShank3 -treated neurons («=19 cells). The data were obtained from seven independent preparations. There are no differences in mEPSC ampli- tudes, growth times or decay times in shShank3 -treated neurons compared to control neurons (**p<0.005; Student's t-test). The data are provided as the average ± SEM.

Figure 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. (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 μΜ and picrotoxin 50 μΜ. In order to isolate the mGluR-mediated long-term potentiation (LTP), aminophosphovaleric acid (APV) 50 μΜ was applied together with DHPG 100 μΜ or applied alone as a control. (A, top) In shCtrl - treated cultures, DHPG 100 μΜ induced a persistent decrease in mEPSC frequencies. (A, bottom) In shShank3 -treated neurons, DHPG 100 μΜ did not induce a decrease in mEPSC frequencies. (B) Averaged data that show the effect of DHPG on mEPSC frequencies in shCtrl -treated neurons (black circles, «=7 neurons) and in shShank3 -treated neurons (grey circles, «=7 neurons). The data are obtained from three independent preparations: mEPSC frequencies within the first 5 minutes of the recording (that is before DHPG application) were set to 100%. (C) The summarizing graph shows changes in the average mEPSC frequencies 30-35 minutes after DHPG application. The differences in the 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 μΜ (+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. (E, bottom) In shShank3 -treated neurons, the numbers of immunoreactive GluRl clusters on dendrite surfaces were reduced and treatment with DHPG did not affect the expression of GluRl on the cell surface. (F) Summary of GluRl distribution on the cell surface in shCtrl- and shShank3 -treated neurons after treatment with DHPG. The chart summarizes data obtained from five preparations. #p<0.05, Student's t-test; *p<0.05, t-test for paired data. The data are provided as the average ± SEM. Figure 5. Restoration of mEPSC frequencies and DHPG-induced ERKl/2 phosphorylation by using CDPPB in hippocampal neuron cultures treated with shShank3, a positive al- losteric modulator of mGluR5. (A) The neurons were infected with the lentivirus on DIV 8 and treated overnight with CDPPB 1 μΜ on DIV 13. mEPSCs were recorded on DIV 14 at a starting potential of -60 mV in the presence of TTX 0.5 μΜ and picrotoxin 50 μΜ. (Α,Β) Summarizing graphs of mEPSC amplitudes (A) and frequencies (B) in the shCtrl + carrier (n= 11 cells), shShank3 + carrier («= 14 cells), and shShank3 + CDPPB («= 14 cells) treatment groups. The data were obtained from four independent preparations. ANOVA analysis pointed out differences in the frequencies of the three groups (p<0.001). (B) mEPSC frequencies are significantly decreased in the shShank3 + carrier group compared to the shCtrl + carrier group (*p<0.05, Dunn's test) and a significant recovery is detected in the shShank3 + CDPPB group (*p=0.05, Dunn's test). The data are shown as the average ± SEM. (C) Hippocampal neurons were infected with shCtrl or shShank3 on DIV 7. After one week, the neurons were treated for 30 minutes as indicated above the panels, then solubilized and analyzed by Western blotting for Shank3, pERKl/2, and ERKl/2 expres- sion, as indicated on the left-hand side of the panels. (C) Average (± SEM) of pERKl/2, normalized against total ERK values and against non-treated values (NT); at least five independent experiments were quantified. After treatment with DHPG, pERKl/2 level was significantly lower in shShank3 -infected neurons compared to shCtrl-infected neurons, *p<0.01 , Student's t-test.

Figure 6. 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. For each mRNA analyzed (Shankl, Shank2 and Shank3), 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, and the right histogram relates to the results obtained with reference to infection with shShank3. 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. For each protein (synaptophysin, Shank3 and PSD-95), 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, and 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.

Figure 7. (A) 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. For each mRNA analyzed (PSD-95, GluRl, GluR2 and mGluR5), 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, and the right histogram relates to the results obtained with reference to infection with shShank3.

Figure 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 μηι), length and width. Over 14 transfected neurons from four independent experiments were measured for each transfection. The numbers, length and width of the dendritic spines were significantly different in shShank3- transfected neurons compared to shCtrl-transfected neurons, **p<0.05, *p<0.01, Student's t-test. Scale bar = 10 μηι.

Figure 9. Shank3 mutations observed in autistic patients do not restore siShank3 -induced deficiencies in mGluR5 pathway. (A) 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). For each treatment condition (NT, DHPG and KC1), 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, and the right histogram relates to values obtained from infection with shShank3+Shank3InsGr. pERKl/2 level was significantly lower after treatment with DHPG in neurons transfected with shShank3, shShank3+shShank3R87Cr and shShank3+shShank3InsGr compared to neurons transfected with shCtrl, *p<0.05, Student's t-test. Scale bar = 20 μιη. Figure 10. Re-insertion of shShank3 -resistant Shank3 into shShank3 -treated hippocampal neurons restores mEPSC frequencies. The neurons were infected with the lentivirus on DIV 8 and transfected on DIV 1 1 with an empty vector (mock) or with a plasmid encoding for shShank3 -resistant Shank3 (Shank3r). On DIV 14, the mEPSCs were recorded at a starting potential of -60 mV in the presence of TTX 0.5 μΜ and picrotoxin 50 μΜ. (Α,Β) Summarizing graphs of mEPSC amplitudes (A) and frequencies (B) in groups treated with shCtrl + mock (n= 12 cells), shShank3 + mock («= 11 cells) and shShank3 + shShank3r (n= 14 cells). The data are obtained from four independent experiments. ANOVA analysis pointed out differences in the frequencies of the three groups (pO.01). (B) mEPSC fre- quencies are decreased in neurons treated with shShank3 + mock (*p<0.05 Dunn's test) and are restored in cells treated with shShank3 + Shank3r (*p<0.05 Dunn's test). The data are provided as the average ± SEM.

Figure 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 μΜ 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 μΜ 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. In each of the panels B-E, the curve marked by an "a" relates to shCtrl and the one marked by a "b" relates to shShank3. There were no differences between shCtrl- and shShank3 -treated groups in baseline activity. Therefore, the values of parameters estimated under the baseline condition were set to 100% for each culture. Two-way ANOVA analysis with repeated measures (DHPG concentrations of 1 μΜ, 10 μΜ and 100 μΜ) revealed a significant effect of DHPG (pO.01), but not of shShank3 (p>0.1) or shShank3 x DHPG (p>0.8) on the mean firing rate (B). Significant effects of DHPG (p<0.01) and shShank3 (p=0.05) on the mean bursting rate were detected in (C), and significant effects of RNAi x DHPG (p<0.05) on the intra-burst firing rate (D) and % of out-burst spikes (E) were detected. *p<0.05, **p<0.01, Holm-Sidak post hoc t- test, significant differences between shCtrl- (n = 10 cultures) and shShank3 -treated cultures (n = 7 cultures).

EXPERIMENTAL SECTION

Materials and Methods

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/mm²) and medium density (150-200 cells/mm²) 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 μΜ DHPG (group I metabotropic glutamate receptor agonist), 100 μΜ NMD A, or 50 mM KC1 at 15 DIV for 30 minutes. To reduce the endogenous synaptic activity, 2 μΜ tetrodotoxin was added to the cultures 12 hours before stimulation. For the experiments with CDPPB, neurons were treated for 12 hours with 100 nM or 1 μΜ CDPPB before DHPG stimulation.

Western blotting and antibodies

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). For the quantification of the immunoblot signals, 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. The following antibodies and dilutions were used (source in brackets): 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-actin 1 :1000, and mouse anti- a-tubulin 1 :1000 (Sigma- Aldrich). Immunocytochemistrv

For immunostaining, 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. The following antibodies and dilutions were used (source in brackets): 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).

RNA interference and plasmids For plasmid-based RNA inhibition, 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 μg 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.

Confocal images of 1024 ^χ 1024 pixels were obtained with a LSM 510 Meta confocal microscope (Carl Zeiss, a gift from Fondazione Monzino) and a ^χ63 objective (1,4 numerical aperture) with a sequential acquisition. Each image was a Z-series projection of 7-15 images, each averaged 2 to 4 times, and taken at 0.4-0.7-μηι depth intervals. Morphometric measurements were made using MetaMorph image analysis software (Universal Imaging). A few individual dendrites were selected randomly and their spines were traced manually. The maximum length and head width of each spine were measured and archived automatically.

To study ERKl/2 phosphorylation, neurons were transfected with the pEGFP, Shank3r, Shank3R87Cr, or Shank3InsGr vectors alone or in combination with Shank3 shRNA at a ratio of 1 :2. Cell bodies were manually traced using MetaMorph software on the GFP channel. The average intensity of the fluorescent signal obtained with antibodies against pERKl/2 in the transfected neurons was divided by the average intensity of the pERKl/2 signal in adjacent untransfected neurons. Cell cultures for patch clamp recordings

Primary cultures of murine hippocampal neurons were prepared as described in Dityatev, A. et al., 2000, Neuron, 26:207-217. Hippocampi obtained from 1 to 3-days old C57BL6/J mice were dissected at 4°C and the neurons were collected by enzymatic digestion with trypsin and mechanical disruption. The cells were then plated at a density of 300/mm² in neurobasal-A medium supplemented with 5 μg/ml gentamicin, 2% B27 supplement, 25 μg/ml FGF2, and 0.5 mM L-glutamine (all from Invitrogen) onto 18 mm-diameter round glass coverslips (Menzel-Glaser) coated overnight with 100 μg/ml poly-L-lysine (Sigma- Aldrich) and 40 μg ml laminin (Sigma- Aldrich). The cultures were maintained at 37°C in a humidified incubator with 95% 0₂ and 5% C0₂. Beginning from day 3 in culture, the medium was supplemented with 0.5 μΜ AraC (Sigma-Aldrich) to prevent glial cell proliferation. The medium was changed twice a week. Infection and transfection of neurons

Primary hippocampal neurons were infected on DIV 6-8 with lentivirus expressing GFP (shCtrl) or GFP plus shRNA to knock down the expression of Shank3 (shShank3) and used for experiments performed on DIV 13-15. For the rescue experiments, the infected neurons were transfected on DIV 11 with pcDNA3 (Mock) or shShank3 -resistant Shank3 (Shank3r). A vector expressing a red fluorescent protein, tdTomato, was co-transfected in these experiments to allow for the identification of transfected neurons. A modification of the calcium phosphate precipitation method was used for the transfection (Jiang M et al., Nat Protoc 2006; l(2):695-700). Briefly, 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% C0₂. 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. For pharmacological rescue of mEPSC frequencies, the allosteric modulator of mGluR5 (CDPPB, 1 μΜ, 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. Whole cell recordings from hippocampal cultures

Whole cell recordings from pyramidal-like neurons were obtained as described in Moult PR, et al, J Neurosci 2006; 26(9): 2544-2554. Electrodes with a resistance in the range of 3-6 MOhms were filled with a solution that contained 130 mM CsMeS0₄, 8 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 0.5 mM EGTA, and 10 mM HEPES, pH 7.25. Cells were perfused continuously with HEPES -buffered saline (HBS) of the following composition in mM: 1 19 NaCl, 5 KC1, 2 CaCl₂, 2 MgCl₂, 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 μΜ, Tocris) was applied by addition to HBS. To prevent any contribution of NMD A receptor-dependent LTD and thus elicit a pure mGluR-dependent LTD, 50 μΜ APV (Sigma) was co-applied with DHPG or applied alone as a control. Data were digitized at 10 kHz. Continuous recording of mEPSCs was made using an EPC10 USB patch clamp amplifier and PATCHMASTER software (HEKA Elektronik). Detection and measurements of mEPSCs, which were collected over a 3 -minute period, were performed using Mini Analysis software (Synaptosoft, Leonia, NJ) after filtering traces at 1 kHz and using a detection threshold of 6 pA {i.e. above 4 times the standard deviation of baseline noise) and visual verification of all detected events. Only cells with a holding current less than -100 p A were analyzed.

Immunofluorescence staining and imaging of hippocampal neurons To estimate cell surface expression of the GluRl subunit of AMPA receptors and its down- regulation by DHPG, untreated or DHPG-treated cultures were briefly washed with PBS and incubated with an antibody against an extracellular epitope of the GluRl subunit (Alomone Labs, agc-004, 16 μg/ml) for 15 minutes at 37 °C. For treatment, either 100 μΜ DHPG + 50 μΜ APV or 50 μΜ APV alone were added to the culture medium for 10 minutes at 37°C. After application of the anti-GluRl antibody, cultures were fixed with 4% formaldehyde in PBS for 20 minutes at room temperature (RT, 20-22°C). Anti-rabbit Alexa 546-conjugated antibody dnvitrogen was applied under non-permeabilizing conditions for 1 hour at RT. The subsequent experiments and analyses were carried out blindly in that the experimenter didn't know which culture had been treated with DHPG and which with the carrier. For measurements of the synaptic surface, all images were collected using a Leica TCS SP5 confocal microscope and a *60 oil immersion objective at 1024 x 1024 pixel resolution. The quantitative analysis was performed using a Z-series projection of five images taken at 0.8-μιη depth intervals. All analyses were performed using NIH ImageJ software. 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-μιη 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 μιη diameter, 200-μιτι center to center interelectrode distance) arranged in an 8 ^χ 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). To reduce thermal stress to the cells during the experiment, 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. 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 μΜ, 10 μΜ e 100 μΜ 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. Data analysis was performed off-line using a custom software, SPYCODE, developed in MATLAB© (The Mathworks, Natick, MA, USA) (Bologna LL et al., Neuroscience 2010; 165(3):692-704); this software comprises a series of tools for the processing of multichannel neural recordings. The data were imported into MATLAB from mcd files (MCS format), and the spikes were detected using the Precise Timing Spike Detection (PTSD) algorithm (Maccione A et al., J Neurosci Methods 2009; 177(1 ):241-249). 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. Following the 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.

Results

To understand the role of Shank3 in synapse formation and function, Shank3 expression was knocked down using RNA interference. Expressed via a lentiviral vector, 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. It is interesting to note that 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).

It has been proposed that 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). In shShank3 -infected neurons there was a significant reduction of mGluR5 both in the total lysate and in the synaptosomal fraction (mean ± SEM for normalized intensity in the total lysate: 0.58 ± 0.03; in the synaptosomal fraction: 0.37 ± 0.05, p>0.01, Student's t-test). Thus, the level of mGluR5 receptor, which binds directly to. Shank3, was reduced in infected neurons. In the same preparations, the inventors observed no significant difference in the levels of many other proteins that are known to be associated with synapses and/or the PSD, including NMDA and AMPA receptors, PSD-95 and IRSp53 (Figures 1 A and B). Also unchanged were the total levels of GKAP and Homer proteins, which can interact directly with Shank3. The reduction in mGluR5 was not dependent on the decrease in mRNA expression as measured by RT-PCR (Figure 7B). Inhibition of Shank3 expression by shRNA in hippocampal neurons was previously shown to reduce dendritic spine numbers (Roussignol et al., 2005 supra). The reduction in spine numbers is confirmed. The number of spines decreases from 4.9 ± 1.0 for 10-μηι dendrites in GFP-infected neurons to 3.1 ± 0.2 for 10-μπι dendrites in neurons infected with shShank3 (Figure 8A). Spine width and length were significantly reduced and increased, respectively, in neurons infected with shShank3, suggesting that the remaining spines are smaller and more immature (Figure 8B). The activation of group I mGluR receptors can lead to a form of LTD (mGluR-LTD) that requires rapid translation of pre-existing dendritic mRNA and involves several signaling molecules including ERK1/2 and CREB. Therefore, the inventors measured ERK1/2 and CREB phosphorylation in response to application of DHPG, a specific agonist of group I metabotropic glutamate receptors. Stimulation with 100 μΜ DHPG induced a specific decrease in ERK1/2 and CREB phosphorylation, whereas NMD A stimulation or KC1 depolarization induced no decrease (Figures 2A-B). The reduction in DHPG-induced ERK1/2 phosphorylation in the shShank3-transfected neurons was rescued by over- expression of shShank3 -resistant Shank3 (Figures 2C-D). The inventors observed that reduction in DHPG-induced ERK1/2 phosphorylation in these neurons could also be rescued by over-expression of full-length mGluR5, but not by over-expression of two shShank3 -resistant Shank3 mutants, designated as Shank3R87Cr and Shank3InsGr, which were found in autistic patients (Figures 9A-B). To further investigate whether Shank3 deficiency affects synaptic transmission at glutamatergic synapses, mEPSCs were recorded in pyramidal-like neurons expressing GFP in shShank3- and shCtrl-treated cultures. Knockdown of Shank3 expression strongly reduced mEPSC frequencies, but neither their amplitude nor their time course were affected (Figures 3A-E). The observed reduction in mEPSC frequencies in shShank3- treated cultures is consistent with the reported increase in mEPSC frequencies when Shank3 is over-expressed in aspiny cerebellar neurons. The reduction in mEPSC frequencies in the subject cultures could be prevented by transfection of shShank3 -treated neurons with the shShank3-resistant form of Shank3, confirming the specificity of shShank3 treatment (Figures 10A-B).

Activation of mGluR5 could lead to a postsynaptic LTD that is mediated by reduced synaptic expression of AMPA receptors. To investigate the functional consequences of Shank3 deficiency for mGluR5 -dependent synaptic plasticity, the inventors stimulated shShank3- and shCtrl-treated cultures with 100 μΜ DHPG. As previously reported for uninfected cultured neurons, DHPG induced LTD (long term depression) in mEPSC frequencies in neurons infected with the control lentivirus. However, knock-down of Shank3 expression impaired the long term depression (LTD). The inventors also found that 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. Furthermore, 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.

To investigate the functional effects of Shank3 deficiency at the neural network level, the inventors performed multisite recordings of neuronal activity using multielectrode arrays (Figure 11 A). No difference between shShank3- and shCtrl-treated cultures was detected under basal conditions (Figure 1 IB). However, DHPG-induced modulation of network activity was more prominent in shCtrl- than in shShank3 -treated cultures. In terms of the total spiking rate in all active channels, there was only a tendency toward lower activity in shShank3 -treated cultures (Figure 1 IB). However, the up-regulation of the number of bursts and spikes out-of-bursts by DHPG was significantly greater in controls than in shShank3 -treated cultures (Figures 1 1C, E). Also, there was no DHPG-induced reduction in the spiking frequency within bursts after knockdown of Shank3 expression. On the whole, these data suggest that signaling of type I metabotropic glutamate receptors is im- paired by knockdown of Shank3 expression, as a result of a decrease in mGluR5 protein at the synapses.

To investigate whether the DHPG-induced decreases in ERK1/2 phosphorylation and mEPSC frequencies were due to reduction of mGluR5 activity in shShank3 -treated cultures, the inventors pharmacologically augmented the activity of mGluR5 using CDPPB as a positive modulator of this receptor. Overnight treatment with CDPPB restored mEPSC frequencies (Figures 5 A, B). Notably, DHPG-induced phosphorylation of ERK1/2 in neurons infected with shShank3 was also found to be rescued by overnight treatment with CDPPB (Figures 5C, D). These data suggest that signaling of type I metabotropic gluta- mate receptors which is altered by the knockdown of Shank3 expression can be restored by treatment with a positive allosteric modulator of the receptor, such as CDPPB. Discussion

The inventors studied the role of Shank3 in synapse function to better understand the pathogenesis of the neurological symptoms of patients affected by Phelan-McDermid syndrome. For this purpose, the inventors specifically knocked down Shank3 expression in neurons, by using shRNA. 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. For example, the over-expression of Shankl induces maturation of dendritic spines without increasing their numbers, whereas the over- expression of Shank3 induces the formation of new synapses and dendritic spines. Shankl targeting to synapses is dependent on the PDZ domain, whereas 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 Zn²⁺ ion binding to the SAM domain. In contrast, Shankl does not bind Zn²⁺ ions, but forms a large structural complex with Homer in the postsynaptic density (PSD).

The specific function of Shank proteins in dendritic spines is 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. 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. Finally, the fact that simple deletions of SHANK3 or SHANK2, but not SHANK1 , have been clearly implicated in the pathogenesis of mental retardation, autism and, more recently, schizophrenia, suggests that the three proteins may have different functions that cannot compensate for each other.

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. Both Shank3 and mGluR5 can be degraded by proteasomes following ubiquitination, suggesting that their interaction can reciprocally modulate their ubiquitination and stabilization. However, the inventors did not find any change in Shank3 protein expression in knock-out mice in which mGluR5 expression had been deleted. Thus, Shank3 might act as a stabilization platform for mGluR5. It has recently been reported that in knock-out mice in which Densin-180 expression had been deleted, mGluR5 protein and signaling are impaired at the synapses. Particularly, 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. As the inventors found that 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.

Despite the observed reduction in mEPSC frequencies and cell surface expression of GluRl -positive clusters in shShank3 -treated neurons, multielectrode recordings did not reveal significant changes in the spiking patterns of neurons under basal conditions. This is not surprising, because the connectivity between cultured neurons is highly redundant. Therefore, despite the differences in synaptic activity observed between control and Shank3 -knockdown neurons in cultures treated with tetrodotoxin, the composite postsynaptic potentials might well exceed the threshold for spike generation in these cells in the absence of tetrodotoxin. Application of DHPG to cultured neurons led to a strong increase in the bursting rate, as previously reported for hippocampal slices. Importantly, DHPG-induced up-regulation of bursting was reduced in Shank3 -knockdown neurons. This result confirms the importance of Shank3 in the regulation of mGluR5 -dependent signaling under physiological conditions, i.e. in the absence of tetrodotoxin. This result also demonstrates the potential importance of Shank3 in mGluR5 activity-induced shaping of neural network activity.

On the whole, the data obtained by the present inventors suggest that 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.

The existence of a link between mGluR-LTD and cognitive disease is suggested by the finding that mGluR-LTD is altered both in the hippocampus and the cerebellum in the mouse model of fragile X. These results led to the development of novel therapeutics for this syndrome that act on mGluR5. In contrast to the findings of the present inventors in Shank3 -knockdown neurons, mGluR-LTD is enhanced in the fragile X syndrome mouse model. 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.

Notably, the use of mGluR5 antagonists or genetic reduction of mGluR5 (in mice that are heterozygous for mGluR5) can reverse multiple phenotypes in mice deficient in FMR1, a gene encoding for FMRP. These phenotypes include increased dendritic spine density and deficiencies in experience-dependent and learning-dependent plasticity in the hippocampus and visual cortex.

Based on these findings, the inventors performed experiments to test whether the reduced mGluR5 activity in Shank3 -knockdown neurons could be rescued by an allosteric agonist of mGluR5, such as CDPPB, and found that both ERK1/2 phosphorylation and mEPSC frequencies were rescued by overnight treatment with CDPPB. CDPPB is able to penetrate the brain and reverse amphetamine-induced locomotor activity and prepulse inhibition deficiencies in rats, two models that are sensitive to antipsychotic drug treatment. These results demonstrate that allosteric modulation of mGluR5 produces behavioral effects and suggest that such a modulation is a feasible approach for increasing mGluR5 activity in vivo.

In conclusion, these findings open new possibilities for the pharmacological treatment of patients affected by Shank3 gene deletions and mutations, particularly the 22ql3 deletion that causes Phelan-McDermid syndrome.

Claims

1. A positive allosteric modulator of mGluR.5, for use in the therapeutic treatment of Phelan-McDermid syndrome.

2. The positive allosteric modulator of mGluR5 for use according to claim 1, for the therapeutic treatment of the cognitive dysfunctions of Phelan-McDermid syndrome.

3. The positive allosteric modulator of mGluR5 for use according to claim 1 or 2, which is selected from the group consisting of ADX-47273, CPPHA, VU-29, VU-36, VU-1545,

DFB (1 -(3-fluorophenyl)-N-((3-fluorophenyl)methylideneamino)methanimine) and CDPPB (3-cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide) and any combination thereof.

4. The positive allosteric modulator of mGluR5 for use according to claim 3, which is CDPPB (3-cyano-N-(l,3-diphenyl-l-H-pyrazol-5-yl)benzamide).

5. The positive allosteric modulator of mGluR5 for use according to any of claims 1 to

4. wherein the therapeutic treatment comprises administering to a patient affected by Phe- lan-McDermid syndrome a therapeutically effective amount of the positive allosteric modulator of mGluR5 which is comprised within the range of 0.1 to 100 mg/kg body weight.

6. The positive allosteric modulator of mGluR5 for use according to claim 5, wherein the patient is a human being.

7. A pharmaceutical composition comprising a positive allosteric modulator of mGluR5 and optional pharmaceutically acceptable excipients, carriers and/or diluents, for use in the therapeutic treatment of Phelan-McDermid syndrome according to any of the preceding claims.