WO2014173900A1 - Method and pharmaceutical composition for use in the treatment of epileptic seizures - Google Patents

Abstract

The present invention relates to a compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression for use in prevention and or treatment of epileptic seizures.

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR USE IN THE

TREATMENT OF EPILEPTIC SEIZURES

FIELD OF THE INVENTION:

The present invention relates to a compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression for use in prevention and or treatment of epileptic seizures.

BACKGROUND OF THE INVENTION:

The most devastating neurological complications of tuberous sclerosis complex (TSC) is therapy-refractory epilepsy ranging from focal seizures to infantile spasms. In the brain, TSC has been characterized by the presence of malformative lesions, the tubers. Tubers induce a derangement of the cortical lamination and are histologically characterized by dysplastic neurons and giant cells, displaying aberrant immature properties. The inventors have hypothesized that misplaced neurons keep immature properties (Ben-Ari, 2009) and that therefore neurons that generate aberrant patterns and seizures may be blocked by drugs that selectively block aberrant channels.

SUMMARY OF THE INVENTION: The inventors show that the NR2C/D subunit of NMDARs is up-regulated in TSC and is an obvious putative candidate to explain the hyperexcitability and hypersynchronisity of TCS networks. They have tested selective antagonists of NR2C/D subunit of NMDARs on an approved genetic mice model of tuberous sclerosis complex for their potential antiepileptic properties. They show that these antagonists are efficient in vitro and in vivo as they reduce/ block the seizures. In addition, in parallel preliminary experiments, the inventors discovered that slices obtained from resections in TSC patients also showed increased expression of the NR2C subunit and that NMDAR-mediated currents were blocked by the same selective antagonist of NR2C/D subunit of NMDARs. These observations suggest that these subunits may play a direct important role in seizure generation in human and as such they can provide a novel antiepileptic strategy.

Thus, the invention relates to a compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression for use in prevention and or treatment of epileptic seizures.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, the term "NR2C/D receptor" also known as GluN2C/D denotes a subunit of NMDA receptors. NMDA receptors are tetrameric assemblies of two glycinebinding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four subtypes (NR2A, NR2B, NR2C, NR2D). NR2C/D is well known see for example (Thompson et al, 2000).

As used herein, "Tuberous sclerosis" or "Tuberous scelrosis complex (TSC)" denotes a multi-system genetic disease that causes non-malignant tumors to grow in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. A combination of symptoms may include seizures, developmental delay, behavioral problems, skin abnormalities, lung and kidney disease, neurological syndromes like epilepsy and autism spectrum disorders. TSC is caused by a mutation of either of two genes, TSC1 and TSC2, which encode for the proteins hamartin and tuberin respectively. These proteins act as tumor growth suppressors, agents that regulate cell proliferation and differentiation.

As used herein, the term "epileptic seizure" denotes a transient symptom of "abnormal excessive or synchronous neuronal activity in the brain often associated with a variety of clinical signs.

As used herein, the term "treating" or "treatment", denotes reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

Compounds and uses thereof

A first object of the invention relates to a compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression for use in prevention or treatment of epileptic seizures.

In one embodiment, epileptic seizures are developmental epilepsies, infantile epilepsies, early epilepsy or adult epilepsies.

In another embodiment, epileptic seizures are epilepsy associated with tuberous sclerosis.

In another embodiment, epileptic seizures are epilepsy associated with focal cortical dysplasia.

In a particular embodiment, the compound according to the invention may be used in prevention or treatment of epileptic seizures in tuberous sclerosis.

In another particular embodiment, the compound according to the invention may be used in prevention or treatment of epileptic seizures caused by genetic mutations in TSCs.

In another embodiment, the compound according to the invention may be used in the prevention or the treatment of autism spectrum disorders related to TSC In a particular embodiment, the compound according to the invention may be used in prevention or treatment of epileptic seizures associated with mTORpathies.

As used herein "mTORpathies" denotes for example neurological diseases like autism, neurodegenerative disease, migration disorders (e.g. neuronal migration disorders) or developmental disorders. mTORpathies also denotes disorders in aged people.

As used herein, the term "mTOR" for "mammalian Target Of Rapamycin" denotes a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. In a preferred embodiment, said compound according to the invention is a NR2C/D receptor antagonist.

In one embodiment, said NR2C/D antagonist may be a low molecular weight antagonist, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In one embodiment, the antagonist may bind to NR2C/D receptor and block the binding of other compound on NR2C/D receptor.

In a particular preferred embodiment, the antagonist of NR2C/D receptor is the UBP141.

In a particular preferred embodiment, the antagonist of NR2C/D receptor is the DQP1105 (see for example Acker, T.M. et al, 2011). Antagonists of NR2C/D are well known in the state of the art (see for example, Koller et al, 2010, Expert Opin. Ther. Patents).

In a particular embodiment, the compound according to the invention may be quinazolin-4-one derivatives (see for example Mosley et al, 2010).

In a particular embodiment, the compound according to the invention may be the phencyclidine (see for example Hagino et al, 2010).

In a particular embodiment, the compound according to the invention may be the CGS 19755 (see for example Kinarsky et al, 2005).

In a particular embodiment, the compound according to the invention may be piperazine-2,3-dicarboxylate derivatives (see for example Costa et al, 2009). In a particular embodiment, the compound according to the invention may be the 1- (phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid (see for example Feng B. et al, 2004). In another embodiment, NR2C/D receptor antagonist of the invention may be an anti-

NR2C/D receptor antibody which neutralizes NR2C/D receptor or an anti-NR2C/D receptor fragment thereof which neutralizes NR2C/D receptor.

Antibodies directed against NR2C/D receptor can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against NR2C/D receptor can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-NR2C/D receptor single chain antibodies. NR2C/D receptor antagonists useful in practicing the present invention also include anti-NR2C/D receptor antibody fragments including but not limited to F(ab')₂ fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to NR2C/D receptor.

Humanized anti-NR2C/D receptor antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non- human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non- human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of NR2C/D receptor are selected.

In still another embodiment, NR2C/D receptor antagonists may be selected from aptamers. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).

Then, for this invention, neutralizing aptamers of NR2C/D receptor are selected.

In a preferred embodiment, the compound according to the invention is an inhibitor of the NR2C/D receptor expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of NR2C/D receptor gene expression for use in the present invention. NR2C/D receptor gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that NR2C/D receptor gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of NR2C/D receptor gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of NR2C/D receptor mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of NR2C/D receptor gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing NR2C/D receptor. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siR A or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Another object of the invention relates to a method for preventing or treating a epileptic seizures by administering to a subject in need thereof a therapeutically effective amount of compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression as described above.

In one aspect, the invention relates to a method for preventing or treating an epileptic seizure comprising administering to a subject in need thereof a therapeutically effective amount of a NR2C/D receptor antagonist as above described.

In another aspect of the invention, the invention relates to a method for preventing or treating epilepsy associated with tuberous sclerosis comprising administering to a subject in need thereof a therapeutically effective amount of a NR2C/D receptor antagonist as above described Compounds of the invention may be administered in the form of a pharmaceutical composition, as defined below.

Preferably, said compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat/reduce and/or prevent epileptic seizures and associated syndromes.

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Compounds according to the invention may be used for the preparation of a pharmaceutical composition for use in prevention or treatment of epileptic seizures.

Hence, the present invention also provides a pharmaceutical composition comprising an effective dose of an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression, preferably a NR2C/D receptor antagonist, according to the invention. Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Screening methods:

Another object of the invention relates to a method for screening a compound which antagonises the NR2C/D receptor.

In particular, the invention provides a method for screening a NR2C/D receptor antagonist for use in prevention or treatment of epileptic seizures. For example, the screening method may measure the binding of a candidate compound to NR2C/D receptor, or to cells or membranes bearing NR2C/D receptor or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the receptor with a labelled competitor (e.g., antagonist).

Furthermore, screening methods may test whether the candidate compound results in a signal generated by an antagonist of NR2C/D receptor, using detection systems appropriate to cells bearing the receptor.

In a particular embodiment, the screening method of the invention comprises the step consisting of:

a) providing neurons expressing NR2C/D receptor on their surface:

b) incubating said cells with a candidate compound ;

c) determining whether said candidate compound binds to and antagonises NR2C/D receptor; and

d) selecting the candidate compound that binds to and antagonises NR2C/D receptor. In one embodiment, the NR2C/D receptor used in the screening method may be its orthologs and derivatives.

In general, such screening methods involve providing appropriate cells which express NR2C/D receptor, its orthologs and derivatives thereof on their surface. In particular, a nucleic acid encoding NR2C/D receptor may be employed to transfect cells to thereby express the receptor of the invention. Such a transfection may be accomplished by methods well known in the art.

In a particular embodiment, cells are selected from the group consisting of glial cells, neuronal cells, neurones, transfected cell lines for investigations or renal cells of any species (mouse, human ... ) .

The screening method of the invention may be employed for determining an antagonist by contacting such cells with compounds to be screened and determining whether such compound antagonises the receptor. According to a one embodiment of the invention, the candidate compound of may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds.

The candidate compound may be selected from the group of (a) proteins or peptides,

(b) nucleic acids and (c) organic or chemical compounds (natural or not). Illustratively, libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163. Further illustratively, the candidate compound may be selected from the group of antibodies directed against NR2C/D receptor.

Such the method may be used to screen NR2C/D receptor antagonists according to the invention.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Characteristics of spontaneous seizures in Tscl+/- mice. Cumulative probabilities of maximal amplitudes of seizures for L2/3 and L4 (upper left) and duration (upper right). Seizure durations were the same for all layers, data for L2/3 are shown. Bottom: Relative integral power of θ-, δ-, α-, β-, γ- and fast ripple (FR) band components of EEG in L2/3 and L4 revealed by Fourier transform analysis.

Figure 2: Layer- specific up-regulation of GluN2B and GluN2C/D subunits containing NMDA receptors in Tscl+/- mice, (a) Summary data for normalized charges of sEPSC (pooled data sets) in L2/3 and L4 in Tsclwt and in Tscl+/- mice. Two-sample t-tests were performed on the pooled data for each group and condition, (b) Summary data for the effects of UBP141 (10 μΜ) and Ro25-6981 (1 μΜ) on normalized charges of sEPSC in L2/3 in Tsclwt and in Tscl+/- mice. Two-sample t-tests were performed on the pooled data for each group and condition, (c) The same as in (b), but for L4.

Figure 3: NMDARs in fast-spiking interneurons are not affected by UBP141 in Tscl+/- mice. Summary data for the effects of UBP141 and Ro25-6981 on normalized charges of sEPSC in L4 fast-spiking interneurons.

Figure 4: Relative expression of mRNAs encoding different NMDAR subunits in cortex of Tscl+/- mice, (a) Quantitative RT-PCR showing the relative expression mRNAs encoding different NMDAR subunits in the cerebral cortex of wild type mice at PI 6. HPRT and Cyclophilin-A were used for normalization. Results are mean ±SEM (n=5 mice for each group), (b) Relative expression of NMD A receptor subunits in Tscl+/- mice compared to wild-type mice. Fold change expressions for each receptor were calculated as: (mRNA Tscl+/- - mRNA Tsclwt)/mRNA Tsclwt.

Figure 5: Single-cell heterozygote or homozygote Tscl knockout induce up- regulation of slow UBP141 sensitive component of NMDA receptor mediated synaptic transmission, (a) Summary data for normalized charges of sEPSC in Tsclflx/wt ; pCAG- mRFP, Tscl+/- mice in Tscl flx/mut; Cag-Cre-GFP, Tsclflx/wt ; Cag-Cre-GFP mice and in Tsc 1 flx/wt ; Cag-Cre-GFP in the presence of UBP 141.

Figure 6: Up-regulation of GluN2C subunit of NMDARs in human postsurgical tissue of TSC patients, (a) Differential expression of NMDA receptor subunits in human brain tissue from normal individuals and TSC patients. Left panel: Quantitative RT-PCR showing the relative expression mRNAs encoding different NMDAR subunits in control samples from human fetal and adult cerebral cortex, β-actin and GAPDH were used for normalization. Error bars represent ±SEM. Right panel: Fold change expressions for each receptor were calculated relative to the fetal brain as follows: (mRNA patients - mRNA fetal control)/mRNA fetal control, (b) Summary data for the effects of 10 μΜ UBP 141 on amplitudes, normalized and total charges of the sEPSC recorded from human postsurgical tissue (pooled data sets). Two- sample t-tests were performed on the pooled data for each parameter.

EXAMPLES:

Material & Methods Quantitative RT-PCR. Total RNA was isolated from mouse cerebral cortex and human brain tissue using RNeasy-Plus Mini Kit, according to the manufacturer's protocol (Qiagen). cDNA was synthesized using the Quantitect Reverse Transcription Kit, according to the manufacturer's protocol (Qiagen). Quantitative real time RT-PCR experiments were performed using oligonucleotides specific for mouse HPRT and cyclophilin-A; human Beta- actin and GAPDH and mouse and human NMDA receptor subunits genes (Oligonucleotide sequences are available upon request). Amplification was done using SYBR-Green and chemistry (Roche diagnostics) and Roche amplification technology (Light Cycler 480). All experiments were performed in triplicate. Human fetal brain and adult cerebral cortex mRNAs, used as controls, were purchased from BD Biosciences Clontech (Palo Alto, CA).

Animal slice preparation. Wild type and Tscl+/- mice (P14-P16) were anaesthetized with ether and killed by decapitation in agreement with the European Directive 86/609/EEC requirements. The brain was rapidly removed and placed in an oxygenated ice- cold saline buffer. Transverse 300 μιη-ΐΐιίΰΐί coronal slices were cut using a vibratome (Leica VT1000S; Leica Microsystems Inc., Deerfield, IL) in ice-cold protecting solution oxygenated with 95% 02 and 5% of C02. Prior to recording, slices were incubated in an ACSF solution containing (in mM): 125 NaCl, 3.5 KC1, 1 CaC12, 2 MgC12, 1.25 NaH2P04, 26 NaHC03, and 10 glucose, equilibrated at pH 7.3 with 95% 02 and 5% C02 at room temperature (22- 25°C) for at least 1 h to allow recovery.

Human cortical slice preparation. After resection, the cortical tissue was placed within 30s in ice-cold artificial cerebrospinal fluid (ACSF) slicing solution which contained in (mM): 110 choline chloride, 26 NaHC03, 10 D-glucose, 11.6 sodium ascorbate, 7 MgC12, 3.1 sodium pyruvate, 2.5 KC1, 1.25 NaH2P04, and 0.5 CaC12 -300mOsm and transported to the neurophysiology laboratory, within less than 5 min. Cortical slices (400-900μιη) were prepared in ice-cold slicing solution, and were then transferred to holding chambers in which they were stored at room temperature (20-22°C) in recording ACSF which contained (in mM): 126 NaCl, 3.5 KC1, 1 MgC12, 2 CaC12, 10 D(+)-glucose, 1.20 NaH2P04, 26 NaHC03 (5% C02 / 95% 02).

Electrophysiological recordings from brain slices. Slices were transferred to the recording chamber and perfused with oxygenated recording ACSF at 3 ml/min. Neurons were visualized using infrared differential interference contrast (IR-DIC) microscopy. Whole-cell patch-clamp recordings were performed at room temperature by using either an EPC-9 amplifier and Patch Master software (HEKA Elektronik, Germany) or Multiclamp 700B amplifier (Molecular Devices, USA) and custom-made software based on IgorPro and filtered at 3-10 kHz. Patch pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, USA) and had resistances of 4 to 6.5 ΜΩ when filled with the internal solution of the following composition (in mM): 130 K-gluconate, 10 Na-gluconate, 4 NaCl, 4 MgATP, 4 phosphocreatine, 10 HEPES, and 0.3 GTP (pH 7.3 with KOH). Biocytin (final concentration 0.3-0.5%) was added to the pipette solution to label the neurons from which recordings were obtained.

The series resistance estimated from the amplitude of the initial capacitive transient in response to a 5-mV pulse was 8 to 24 ΜΩ. It was not compensated and was monitored during each experiment. Experiments were terminated if the series resistance changed by more than 15%. Spontaneous EPSCs were recorded for 15 min at -80 mV (the reversal potential for GABAergic currents) and at -50 mV (potential at which the block of NMDAR by magnesium is largely relieved). All recordings were made in normal ACSF (1 mM Mg2+) without any proepileptic pharmacological drug. To minimize potential sampling bias, the pups from at least three deliveries for each condition were studied. Data Analysis and Statistics. The Mini Analysis 6.0.3 software (Synaptosoft Inc.,

Decatur, GA) was used to analyze the kinetics, frequency and amplitude of synaptic events. The threshold amplitude for detecting EPSCs was set at twice the baseline noise (root mean square), and the EPSCs detected by the software were visually inspected to minimize errors. Events that did not show a typical synaptic waveform were rejected manually.

For kinetic analysis, only events that did not show any signs of multiple peaks (i.e., contamination of rise or decay phases by subsequent events) were selected for analysis of the kinetics and for exponential fitting. To quantify the current decay kinetics we used normalized charges of sEPSC (charge transfer measured in the range of 300 ms from peak of the sEPSCs normalized by the peak amplitude). Data are expressed as mean ±S.E.M. of results obtained from various animals. Peak amplitudes and decay time constants were estimated for single sEPSCs and further analyzed on a personal computer (MiniAnalysis; Synaptosoft; and Origin; MicroCal, Northampton, MA). All comparisons were one-tailed t-tests. Statistical significance for cumulative probabilities was estimated using Kolmogorov-Smirnov test. Immunocytochemistry. After the recording session, to visualize and identify the recorded neurons, we visualized the biocytin injected during whole cell recordings. After 24h in paraformaldehyde (3%) at 4°C, the sections were rinsed in PBS and pre-incubated for 1 h in 0.3 % triton (Abcys) in PBS with 5% normal goat serum (NGS) at room temperature. Slices were then incubated in Streptavidin-Cy3 (1 :500) in PBS triton (0.3%) and NGS (5%) during 12 h at 4°C. After thorough rinsing, slices were mounted in fluoromount and coverslipped.

In vivo recordings and data analysis. This study followed the Institut National de la Sante et de la Recherche Medicale guidelines for animal care. All experiments were performed on postnatal days P9-P20 of inbred C57B16 strain of both sexes of Tsclmut/wt (Tscl+/-) mice issued from breading of C57B16 Tsclwt females and Tsclmut/wt males Tsclmut/wt.

Surgery was performed under isoflurane anesthesia. In brief, the skull of the animal was cleaned of skin and periosteum. The skull was covered by glue and dental cement except for a 4-9 mm2 window above the somatosensory cortex from one or two hemispheres. Two plastic bars were fixed to the nasal and occipital bones of the pups head by dental cement. After surgery, animals were warmed, and left for an hour for recovery from anesthesia. During this time mice were connected to multichannel extracellular recording system (A-M systems, Neuronexus) for 8-24 hours. During recordings, the head was fixed to the frame of the stereotaxic apparatus by the attached bars; animals were surrounded by a cotton nest and heated via a thermal pad (36.6°C -37.7°C). A silver chloride reference electrode was placed in the cerebellum or visual cortex.

Electroencephalography (EEG) recordings were performed in non-anesthetized head- restrained Tscl+/- and control Tsclwt mice. 16-site linear silicon probe (100 μιη separation distance between recording sites, Neuronexus Technologies, MI) was placed into the somatosensory cortex using the Paxinos and Franklin atlas (2001) at coordinates: AP=2-2.5 mm, L=2-3 mm; 1.2-1.5 mm depth, to trace the columnar activity at all layers and CA1 zone of hippocampus. The signals were amplified (xlOO) and filtered at 3 kHz using a 16 channel amplifier (A-M systems, Inc), digitized at 10 kHz and saved to hard disk of PC using Axoscope software (Molecular Devices, Sunnyvale, CA, USA). Recordings were analyzed off-line using Clampfit and MATLAB software. In 9 experiments, saline solution (200 μί) n=3 or UBP141 (200μΕ, 75 mg/kg) n=6 was injected intraperitoneally (IP). After the recordings, position of silicone probe was verified visually or by Dil staining of the electrode or in 100 μηι coronal sections from fixed brain. We considered that multiunit activity occurred in epileptic discharges if they appeared in a group of multiple spikes whose amplitude exceeded at least twice the background activity within a period lasting for at least 20 s. The first and last spikes of each discharge were used to define its onset and termination, respectively. For each discharge amplitude was defined as the amplitude of the largest spike of the discharge. During EEG recordings animals were monitored visually to determine behavioral correlates of each electrographic epileptic discharges.

For EEG data analysis raw data were preprocessed using a custom-developed suite of programs in the MATLAB analysis environment. The wide-band signal was downsampled to 1000 Hz and used for local field potential signal. Positive polarity is graphed as up throughout all manuscript.

Local field potentials were analyzed by custom- written, MATLAB based programs. Approximate anatomical location of each recording site was estimated by physical depth within the brain and corresponding age-matched histological assessment of respective layers depth.

Negative epileptic events were detected by the following steps: 1) the LFP signal was bandpass filtered (5-100Hz), 2) the times of negative troughs with amplitude greater than 5 Standard deviations from baseline level were detected from filtered signal, 3) aligned by the times of detected negative epileptic events; the lfp segments from all channels were taken. Note the length of the segment is 200 ms with moment of negative trough at 0.

Current-source density (CSD) analysis across cortical depth was used to eliminate volume conduction and localize synaptic currents. CSD was computed for each recording site according to differential scheme for second derivative and smoothed with a triangular kernel of length 3).

Multisite extracellular recordings from brain slices. Multisite extracellular recordings of spontaneous activity in slices were performed at 30-32 °C using MEAs made up of 60 planar microelectrodes (TiN/SiN, 30 μιη electrode diameter, 200 um pitch) arranged over an 8 x 8 square grid (Multi Channel Systems [MCS], Reutlingen, Germany). Slices were maintained in dishes and perfused with oxygenated recording ACSF. The spontaneous activity was monitored and recorded for 30-120 min, starting 15-20 min after setting slice in the recording chamber at a stable level of activity. After 1200^x amplification (MCS MEA 1060), signals were sampled at 10 kHz using the MCS data acquisition card controlled by the MCS MCRack software. Data were analyzed off-line by using custom software tools specifically developed in MATLAB (The Mathworks, Natick, MA).

Human Subjects. Cortical tissue samples were obtained from 3 TSC epilepsy patients undergoing surgery at the Departments of Pediatric Neurosurgery of Rothschild Foundation (Paris) and Hopital La Timone (Marseille) between January 2011 and October 2012.

Informed consent for the use of postsurgical tissue for research purposes was obtained with protocols approved by the institutionals review boards.

TSC specimens were collected from patients who underwent surgery for medically intractable epilepsy. All patients were clinically diagnosed with TSC and presented a history of epilepsy.

Chemicals: All drugs were prepared as concentrated stock solutions (10-100 mm), stored frozen and then thawed and diluted in ACSF-2 immediately before use. Ro-25697 (aR,pS)-a-(4-Hydroxyphenyl)-P-methyl-4-(phenylmethyl)-l-piperidinepropanol maleate, and DQP1105 5-(4-Bromophenyl)-3-(l,2-dihydro-6~methyl-2-oxo-4-phenyl-3-quinolinyl)-4,5- dihydro-g-oxo-lH-pyrazole-l-butanoic acid were purchased from Tocris Biosciences (Bristol, UK). UBP 141 (2R*,3S^!i:)-l-(Phenanthrenyl-3-carbonyl)piperazine-2,3-dicarboxylic acid was purchased from AbcamBio chemicals. All other chemicals used for electrophysiology were from Sigma (USA).

Treatment with rapamycin: Rapamycin (ready made solution, Sigma- Aldrich, USA, 2.5 mg/mL in DMSO (2.74 mM)) diluted with saline to a final concentration 24 mg*kg-l immediately prior to use. The mice received the rapamycin solution (3 mg*kg-l) or an equal volume of vehicle by intraperitoneal injection once daily for 8 consecutive days. The electrophysiological recordings were performed 24 h after the last administration.

EXAMPLE 1: first results Results

Mice experiments: In animal models of TSC, a second "hit' was focally induced on a heterozygous background for Tscl mutation and resulted in cellular abnormalities reminiscent of tubers, providing support for biallelic gene inactivation in tuber formation. However a second mutation hit in TSC1 or TSC2 is shown to be a rare event in human tubers (Qin et al, 2010).

To characterize functional abnormalities of haploinsufficient Tscl+/- mice, lacking major malformations, in vivo intra-cortical electroencephalography (EEG) recordings in somatosensory SI cortex of head-restrained non-anaesthetized Tscl+/- mice (P9-P20) were performed. Spontaneous recurrent seizures were revealed in 80% of the mice tested (20 out of 25). The seizures started 2-3 hours after onset of EEG recordings and reappeared up to 6 per hour. Ictal discharges were often associated with screaming, oro-facial automatisms, tonic extension of the trunk, and straub tail, followed by quiet behavior (data not shown).

Ictal EEG patterns started as high-frequency low-amplitude activity that progressively evolved to high-amplitude regular polyspike trains involving all cortical layers. Subsequently, there was a disruption of the discharge with a reduction in EEG amplitude in cortex and appearance of high-amplitude rhythmic spike-wave trains in the hippocampus (data not shown). Wavelet analysis showed an increase in high frequency activity during ictal discharges (data not shown). The contributions of theta, gamma and fast ripple bands in power spectrum during discharges were significantly larger in neocortical layer 4 (L4) compared to layer 2/3 (L2/3) (Figure 1, bottom). The amplitude and duration of discharges varied within litters with the mean amplitude of 622±1 μν and mean duration of 60.7±0.3 s, (n (seizures)=128; n (mice)=20, Figure 1, upper panels).

The seizures onset as well as the peaks of averaged population spikes in L4 preceded that in L2/3 (the mean delay for the peaks was 8.0±1.3 ms (n=l l mice)). These observations as well as current source density analysis suggest that epileptic activities are initiated in granular cortical layer then spreading rapidly to supra- and infragranular layers (data not shown). Accordingly, in simultaneous whole-cell recordings from somatosensory cortical excitatory neurons in L4 and L2/3 in Tscl+/- mice coronal slices, where thalamocortical inputs are absent, but most of intralaminar circuitry and horizontal recurrent connections are intact, bursting events during spontaneous synchronous activities occur first in L4 and subsequently in L2/3 neurons with an average delay of 30.4±5.5 ms (65 synchronous bursts were analyzed, data not shown).

L4 is the main collector of sensory information and cortical "hub" of intracolumnal information processing (Feldmeyer 2012). Recurrent activity triggered within the highly interconnected networks of L4 has been suggested to act to selectively amplify and redistribute transient high-frequency thalamocortical inputs. What are the mechanisms underlying the increased integrative capacity of L4 neurons in Tscl+/- mice? NMDARs in L4 have been suggested to play an important role in neuronal integrative properties. Importantly, L4-L4 connection is almost the only intracortical input the L4 spiny stellate cells receive. To monitor the NMDAR-mediated current we performed whole-cell recordings of sEPSCs from L4 spiny stellate cells (SSC) and L2/3 pyramidal neurons (PN) in Tscl+/- mice coronal slices in voltage-clamp mode at -50 mV.

The average decay kinetics of late component in composite sEPSCs reflecting the NMDAR-mediated current was significantly slower in Tscl+/- than that in Tsclwt mice both in L2/3 and L4 neurons (Figure 2a) suggesting an up-regulation of GluN2B, and/or GluN2C/D NMDAR subunits endowed with a slow decay kinetics. Thus, in Tscl+/- mice normalized charges of sEPSC (see Methods) were 1.5±0.1 in L2/3 and 2.2±0.17 in L4 of those in Tsclwt mice (L2/3: n=27 and n=16, p<3* 10-5; L4: n=19 and n=22, p<4* 10-6 for Tscl+/- and Tsclwt mice, respectively). All comparisons were one-tailed t-tests.

To directly determine the involvement of NMDAR subtypes in prolongation of sEPSCs we used specific GluN2B (Ro25-6981) and GluN2C/D (UBP141) antagonists. In L4 SSCs of Tscl+/- mice UBP141, but not Ro25-6981, accelerated sEPSCs decay kinetics, restoring it to the values of Tsclwt mice (Figure 2c) suggesting an increased contribution of GluN2C/D but not GluN2B subunits. In Tscl+/- mice the normalized charge of sEPSC in L4 in the presence of UBP141 (10 μΜ) was 0.63±0.03 of that without UBP141 (n=9, p<2* 10-5). In Tsclwt mice these values were similar in the presence and absence of the drug (0.99±0.12, n=8). In contrast, contribution of both GluN2B and GluN2C/D subunits was increased in L2/3. In the presence of 1 μΜ Ro25-6981 the normalized sEPSC charge in Tscl+/- mice was 0.79±0.05 of control without Ro25-6981 (n=9, p<0.003). In Tsclwt mice this value was 1.03±0.09 (n=6, p>0.15). Similarly, corresponding numbers for UBP141 in L2/3 were 0.8±0.07 of controls without the drug (n=12, p<0.01) and in Tsclwt mice, 1.02±0.07 (n=9, p>0.9) (Figure 2b). Therefore, there is an increased contribution of GluN2C/D subunits in L4, but of both GluN2B and GluN2C/D subunits in L2/3. UBP141 altered neither the amplitude nor the kinetics of AMPAR-mediated sEPCS recorded at -80 mV in L4 SSC in Tscl+/- mice (data not shown). Interestingly, in line with the predominant expression of GluN2A subunits in interneurons, neither UBP141 nor Ro25-6981 altered sEPSCs of L4 interneurons in Tscl+/- mice (Figure 3) suggesting that NMDAR-mediated currents in inhibitory neurons remain intact. To further determine the functional effects of UBP141, we tested its actions on the frequency of AMPAR-mediated sEPSCs. In Tscl+/- mice the frequency of sEPSCs recorded at -80 mV in L4 SSC was not different from that in Tsclwt mice (data not shown). However, in L2/3 PN it was significantly higher than that of Tsclwt mice (data not shown), and was reduced by bath application of UBP141 in Tscl+/- (data not shown), but not in Tsclwt (data not shown) mice. Therefore, in Tscl+/- mice L4 GluN2C/D channels presynaptic to L2/3 PN are also up-regulated increasing activity of the latter.

Up-regulation of the NMDAR subunits was confirmed by quantitative RT-PCR revealing 1.4-fold elevation of GluN2C mRNAs (p<0.005) in the neocortex of Tscl+/- (n=5) compared to age-matched Tsclwt (n=5) mice (Figure 4). Therefore, the contribution of NMDARs with slow kinetics is increased in neocortical L4 and L2/3 excitatory neurons in mice with hap lo insufficient Tscl mutations.

In a recent study, using in utero electroporation and relying on the «second-hit» mutations, Bordey and co-workers generated a Tscl-/- animal model with the hallmark of human TSC, namely the tubers [Feliciano, D. & Bordey, A., 2011]. We used this model to test the impact of «second-hit» mutation on functional up-regulation of slow NMDARs. To do that Tscflx/mut mice (as well as Tsclflx/wt) were injected in utero at E16 embryonic stage with pCag-Cre-GFP constructs to induce, after electroporation, deletion of the floxed Tscl gene in a subset of labeled neurons.

Whole-cell recordings from the labeled cells of Tsclflx/mut;Cag-Cre-GFP and

Tsclflx/wt;Cag-Cre-GFP conditional knock-out mice at -50 mV revealed enhanced contribution of slow UBP 141 -sensitive NMDAR-mediated components in sEPSC, as in non- electroporated heterozygote Tscl+/- mice. Therefore, GluN2C/D-mediated currents are present in both Tscl+/- and «double hit» Tscl-/- cells. Importantly, the extent of slow NMDARs contribution was the same for the electroporated neurons with heterozygote and homozygote Tscl mutation (Figure 5).

The observation that seizures are generated in L4 suggests that the selective blockade of long-lasting GluN2C/D subunits containing NMDAR-mediated currents may have antiepileptic effects. To test this possibility, we used microelectrode array extracellular recordings in acute coronal neocortical slices taken from P15 Tscl+/- mice. Spontaneous discharges lasting for up to 10 seconds were recorded in L2/3 and L4. UBP141 (10 μΜ) selectively reduced the amount of long-lasting epileptiform episodes, without altering the number of interictal bursts (< 500 ms) (data not shown). We next tested the antiepileptic actions of UBP141 in vivo by intraperitoneal (IP) injections of the drug (75 mg/kg) to Tscl+/- mice. In 3 of 6 mice tested the seizures were completely stopped ~40 min after IP injection and in the remaining mice (n=3), there was a seizure free period for 109±40 minutes (data not shown). Importantly, after UBP141 injection a basal activity remained unaltered in particular at gamma frequency band known to be enhanced by common NMDAR antagonists (data not shown). In contrast, recurrent epileptic discharges persisted up to 7 hours in Tscl+/- mice that did not receive the antagonist (data not shown). Therefore, selective antagonist of GluN2C/D containing receptors has in vivo and in vitro antiepileptic actions in Tscl+/- mice.

Human experiments:

To test whether findings obtained in the animal model can be translated to human patients with TSC, we performed studies of human postsurgical tissue. Quantitative RT-PCR performed in the 3 human samples with TSC2 mutation (age at surgery ranged from 8 to 16 months) revealed 20-fold increase of GluN2C mRNA compared to fetal control brains (p<0.001) (Figure 6a). Furthermore, whole-cell patch-clamp recordings in brain slices from the same human TSC specimens showed that sEPSCs recorded from dysplastic neurons in tubers and perituberal regions (data not shown) were significantly curtained by UBP141 (Figure 6b), reinforcing functional implication of GluN2C/D in epilepsy associated with TSC in human patients.

Conclusions:

The principal conclusions of our study are that heterozygote mutation in Tscl gene is sufficient to induce epilepsy in the developing Tscl+/- mouse despite the absence of tubers. Seizures are generated intra-cortically due to an up-regulation of GluN2C/D receptors in recurrent connections between SSC in the granular layer of neocortex and then propagate to other layers. SSC with functionally upregulated long-lasting GluN2C/D receptors become effective "hyperintegrators" of powerful and persistent NMDAR-mediated recurrent excitation. This suggests that in Tscl+/- mice seizure initiation could be triggered by conventional sensory inputs to L4. Indeed, in some mice tactile stimulation of the mouse back induced prominent seizures (data not shown).

Our observations provide the first functional evidence that GluN2C/D receptors are up-regulated in neurons of Tscl+/- mice but also in human patients. Although general antagonists of GluN2A-mediated signaling have failed in preclinical studies, our results suggest that NMDAR-subunit targeted therapy may provide a promising novel therapeutic avenue to treat epileptogenesis in TSC. Importantly, selective antagonists do not affect pro- survival GluN2A-mediated signaling. Given the increased expression of specific NMDAR subunits found in tissue resected from patients with various types of drug-resistant epilepsy, the proposed mechanisms of the intracortical epileptogenesis and NMDAR-subunit targeted therapy may be further extended to other types of epilepsies, including focal cortical dysplasia, the most frequent congenital lesions leading to epilepsy.

EXAMPLE 2: Additional results and new results

1. Spontaneous seizures in Tscl+/- mice

To characterize functional abnormalities of haploinsufficient Tscl+/- mice, lacking major malformations, in vivo intra-cortical electroencephalography (EEG) recordings in somatosensory SI cortex of head-restrained non-anaesthetized Tscl+/- mice (P9-P33) were performed. Spontaneous recurrent seizures occurred in 77% of Tscl+/- mice tested at P9-P18 (26 out of 34), but were not observed in Tsclwt mice (data not shown). The seizures started 2-3 hours after onset of EEG recordings and recurred as often as 6 per hour. Ictal discharges were often associated with screaming, oro-facial automatisms, head tremor, straub tail, and tonic-clonic seizures (data not shown), followed by a quiet behavior (data not shown).

Ictal EEG patterns started as high-frequency, low-amplitude activity that progressively evolved to high-amplitude regular polyspike trains involving all cortical layers. Subsequently, there was a disruption of the discharge with a reduction in EEG amplitude in cortex and appearance of high-amplitude rhythmic spike-wave trains in the hippocampus (data not shown). Wavelet analysis showed an increase in high frequency activity during ictal discharges (data not shown). The contributions of δ, γ and fast ripple bands in power spectrum during discharges were significantly larger in neocortical layer 4 (L4) compared to layer 2/3 (L2/3) (Fig. Id, bottom). The amplitude and duration of discharges varied within litters with a mean amplitude of 615±18 μν and a mean duration of 70.3±5 s, (n=104 seizures; N=20 mice, data not shown).

Interestingly, epileptic phenotype was not observed in Tscl+/- mice at ages older than

P19 (P19-P33, N=10), indicating that this is a developmental insult.

The seizure onset as well as the peaks of averaged population spikes in L4 preceded those in L2/3 (data not shown, mean delay for the peaks was 8.0±1.3 ms, N=l 1 mice). These observations and current source density analysis suggest that epileptic activities in the cortex are initiated in granular cortical layer before spreading to supra- and infragranular layers (data not shown). This was confirmed by cross-correlation analysis of the epileptic activity filtered in its dominant frequency band (Θ, 4-8 Hz) (data not shown), showing maximum cross correlation function for L4 to other layers at positive time-lags. This indicates that the signals in other cortical layers are delayed with respect to L4 (t-test, p<0.01). Qualitatively similar results were obtained in coronal slices of Tscl+/- mouse brains, where intralaminar circuitry and horizontal recurrent connections were intact, but thalamocortical inputs were absent. In simultaneous whole-cell recordings from somatosensory cortical excitatory neurons in L4 and L2/3 spontaneous synchronized bursts were observed first in L4 and subsequently in L2/3 neurons with an average delay of 30.4±5.5 ms (65 synchronous bursts were analyzed, data not shown). Therefore, L4 neurons play a central role in neocortical epileptogenesis in Tscl+/- mice.

2. Layer-specific up-regulation of GluN2B and GluN2C/D-subunits containing NMD A receptors in Tscl+/- mice

L4 is the main collector of sensory information and cortical "hub" for intracolumnal information processing. Recurrent activity triggered within the highly interconnected networks of L4 has been suggested to act to selectively amplify and redistribute transient high-frequency thalamocortical inputs. What are the mechanisms underlying the increased integrative capacity of L4 neurons in Tscl+/- mice? As NMDARs play an important role in L4 neuron integrative properties, we next examined whether slow NMDAR-mediated signaling was altered in Tscl+/- mice.

The L4-L4 connections are almost the only intracortical synaptic input that L4 spiny neurons receive. This allows estimation of the contribution of the slow NMDA component in isolated L4-L4 connections by measuring spontaneous activity from spiny stellate neurons. To monitor the NMDAR-mediated current, we performed whole-cell recordings of spontaneous excitatory postsynaptic currents (sEPSCs) from L4 spiny stellate cells (SSC) and L2/3 pyramidal neurons (PN) in coronal brain slices from Tscl+/- mice in voltage-clamp mode at - 50 mV (data not shown). The decay kinetics of the composite sEPSCs was significantly slower in Tscl+/- than that in Tsclwt mice both in L2/3 and L4 neurons (data not shown), suggesting an increased contribution of NMDAR-mediated current and an up-regulation of GluN2B, and/or GluN2C and GluN2D NMDAR subunits endowed with slow decay kinetics. To quantify alterations in sEPSCs decay, we measured charge transfer normalized by the peak amplitude. In Tscl+/- mice normalized charges of sEPSC were 1.5±0.1 in L2/3 and 2.55±0.16 in L4 of those in Tsclwt mice (L2/3: n=27 and n=16 neurons; L4: n=34 and n=29 neurons, for Tscl+/- and Tsclwt mice, respectively; ANOVA, p<3.2* 10-14; Fisher LSD test Tsclwt vs Tscl+/- in L23 p<3* 10-4; in L4 p <1 * 10-13). Bi-exponential weighted time constants of sEPSC decay (Tw) were in Tsclwt mice 7.5±0.6 for L2/3 and 7.4±0.4 for L4 and in Tscl+/- mice 13.28±0.97 in L2/3 and 16.76±1.19 for L4 (ANOVA, p<2.13* 10-11; Fisher LSD test Tsclwt vs Tscl+/- in L23 p<3.4* 10-4; in L4 p <7.4* 10-14). Corresponding amplitudes of sEPSC in Tscl+/- mice were not significantly different from those in Tsclwt mice for L4 (ANOVA, p<0.9) however slightly increased in L2/3 (data not shown).

To directly determine the involvement of NMDAR subtypes in prolongation of sEPSCs, we used specific GluN2B (Ro25-6981) and GluN2C/D (UBP141 and DQP1 105) antagonists (data not shown). In L4 SSCs of Tscl+/- mice UBP141 and DQP1105, but not Ro25-6981, accelerated sEPSCs decay, restoring it to the values of Tsclwt mice (data not shown), suggesting an increased contribution of GluN2C/D but not GluN2B subunits. In Tscl+/- mice the normalized charge of sEPSC in L4 in the presence of UBP141 (10 μΜ) was 0.61±0.07 (n=10, p<4* 10^"4, t-test) and in the presence of DQP1105 (10 μΜ) was 0.48±0.04 (n=12, p<2* 10^~7, t-test) of that without drugs. In Tsclwt mice, sEPSCs decay kinetics were not affected by either drug (the normalized charge values were similar in the presence and absence of the drugs: 1.03±0.13, n=9, p>0.75 for UBP141 and 1.06±0.07, n=12, p>0.4 for DQP1105, t-test).

In addition, we performed recordings of miniature EPSCs (mEPSCs) in L4 cells in the presence of tetrodotoxin (TTX, 1 μΜ). Similarly to sEPSCs, the average decay kinetics of late component in composite mEPSCs was significantly slower in Tscl+/- than that in Tsclwt mice (data not shown). Thus, in Tscl+/- mice averaged normalized charge of mEPSC was 2.58±0.18 in L4 of those in Tsclwt mice (n=10 and n=9 neurons, for Tscl+/- and Tsclwt mice, respectively; ANOVA, p<1.4* 10-8; Fisher LSD test Tsclwt vs Tscl+/- p<2.4* 10-8). Corresponding amplitudes of mEPSC for all sets were not significantly different (ANOVA, p<0.9, data not shown). In Tscl+/- mice the normalized charge of mEPSC and Tw in L4 in the presence of DQP1105 (10 μΜ) were 0.45±0.07 (n=8, p<1.7* 10-4, t-test) and 0.55 ±0.03 (n=9, p<3.2* 10-5, t-test) respectively of those without drugs. In Tsclwt mice mEPSCs decay kinetics were not affected by DQP1105 (the normalized charge and Tw in the presence of

DQP1105 were 0.96±0.14 (n=7, p>0.75, t-test) and 1.13 ±0.13 (n=8, p>0.48, t-test) respectively of those without drug. In contrast to data in L4, contribution of both GluN2B and GluN2C/D subunits was increased in L2/3 of Tscl+/- mice. In the presence of 1 μΜ Ro25-6981 the normalized sEPSC charge in Tscl+/- mice was 0.79±0.05 of control without Ro25-6981 (n=9, p<0.005, t-test). In Tsclwt mice this value was 1.03±0.09 (n=6, p>0.75, t-test). Similarly, corresponding numbers for UBP141 in L2/3 were 0.8±0.07 of controls without the drug (n=12, p<0.02) in Tscl+/- mice and 1.02±0.07 (n=9, p>0.8, t-test) in Tsclwt mice, (data not shown). However, the interpretation of UBP141 effects in L2/3 is complicated by the facts that at concentration of 10 μΜ it partially affects also GluN2B receptors (data not shown). It should be specifically noted that the amplitudes of sEPSC in the presence of antagonists were not significantly different from control values for all experimental sets (data not shown).

Therefore, there is an increased contribution of GluN2C/D subunits in L4, but of both GluN2B and GluN2C/D subunits in L2/3 in Tscl+/- mice when compared to naive Tsclwt mice.

To determine whether up-regulation of GluN2C/D-NMDAR component in L4 is a direct consequence of enhanced mTOR signaling caused by Tscl inactivation, we performed experiments in Tscl+/- mice chronically treated with the mTOR inhibitor rapamycin (see Methods). Recordings of sEPSC from SSC in L4 in rapamycin-treated Tscl+/- mice (P14-16) showed the absence of UBP 141 -sensitive component (data not shown). In rapamycin-treated Tscl+/- mice the normalized charge of sEPSC in SSC in the presence of UBP141 (10 μΜ) was 1.05±0.11 (n=12, p>0,6, t-test) of that without the drug. Furthermore normalized charge of sEPSC in rapamycin-treated Tscl+/- mice was not significantly different from that of vehicle-treated Tsclwt mice (14.01±1.15 (n=14) vs 13.34±1.69 (n=10), t-test p>0.75) (data not shown). This finding indicates a crucial role of mTOR signaling in up-regulation of GluN2C/D-containing NMDARs in Tscl+/- mice.

Interestingly, that in L4 fast-spiking interneurons, neither UBP 141 nor Ro25-6981 altered sEPSCs decay in both Tsclwt and Tscl+/- mice (data not shown), suggesting that NMDAR-mediated currents in inhibitory neurons remain intact.

To further determine the functional effects of UBP 141 in Tscl+/- mice, we tested its actions on the amplitude and frequency of AMPAR-mediated sEPSCs. UBP141 altered neither the amplitude nor the kinetics of AMPAR-mediated sEPCS recorded at -80 mV in L4 SSCs (data not shown). The frequency of sEPSCs recorded at -80 mV in L4 SSCs was not different from that in Tsclwt mice. However, in L2/3 PN it was significantly higher than that of Tsclwt mice (data not shown), and was reduced by bath application of UBP141 in Tscl+/- (data not shown), but not in Tsclwt (data not shown) mice. Therefore, in Tscl+/- mice L4 GluN2B/C/D channels presynaptic to L2/3 PN could contribute to increase activity of the latter.

Up-regulation of the slow NMDAR subunits was confirmed by quantitative RT-PCR revealing 2.4-fold elevation of GluN2C mRNAs (p<0.005) in the neocortex of Tscl+/- mice (N=5) compared to age-matched Tsclwt (N=5) mice (data not shown). Therefore, the contribution of NMDARs with slow kinetics is increased in neocortical L4 and L2/3 excitatory neurons in mice with hap lo insufficient Tscl mutations.

Similar alterations in sEPSC kinetics were observed in "double hit" Tscl mutant mice. In a recent study, using in utero electroporation and relying on the «second-hit» mutation concept, Bordey and co-workers generated a Tscl-/- animal model with the hallmark of human TSC, namely the tubers. We used this model to test the impact of «second-hit» mutation on functional up-regulation of slow NMDARs. To do that Tsclflx/mut mice (as well as Tsclflx/wt) were injected in utero at E16 embryonic stage with a pCAG-Cre construct to induce, after electroporation, deletion of the floxed Tscl gene in a subset of neurons. These neurons were also labeled with a commonly used fluorescent reporter - monomeric red fluorescent protein (mRFP) for visualizing and detecting cells with Tscl loss.

Whole-cell recordings in neocortical slices from mRFP+ neurons of Tsclflx/mut;pCAG-Cre and Tsclflx/wt;pCAG-Cre conditional knock-out mice (hereafter referred to as Tscl null and Tscl hap lo neurons, respectively) at -50 mV revealed enhanced contribution of slow UBP 141 -sensitive NMDAR-mediated components in sEPSC compared to control Tsclflx/wt; mRFP, in the same manner as in non-electroporated heterozygote Tscl+/- mice. Therefore, GluN2C/D-mediated currents are present in both Tsclhaplo and «double hit» Tsclnull neurons. Importantly, the extent of slow NMDARs contribution was the same for the electroporated neurons with heterozygote and ho mo zygote Tscl mutation (data not shown).

3. Slow decay kinetics of NMDAR-mediated signal determines temporal integration of excitatory transmission of high frequency inputs

To test temporal summation at γ band frequency we performed simultaneous whole- cell recordings from pairs of synaptically coupled spiny stellate cells in L4 of somatosensory cortex in slices from Tscl+/- and Tsclwt P14-16 mice. The majority of interconnected excitatory neurons were spiny stellate cells with an asymmetrical dendritic arborization largely confined to L4 (data not shown). In concordance with the with the slower NMDAR- mediated component of synaptic transmission in Tscl+/- mice, recordings revealed a significantly higher extent of temporal integration compared to Tsclwt mice when measuring EPSCs evoked by stimulation of presynaptic cells with a train of action potentials (data not shown). Normalized charge transfer of the train-EPSCs estimated from normalized by the amplitude of the first peak currents was 0.093±0.004 in Tscl+/- (n=5) and 0.050.±0.006 (n=7) in Tsclwt mice, respectively (p<0.0005, two-tails t-test). Furthermore, recordings in Tscl+/- mice demonstrated significantly increased contribution of UBP 141 -sensitive NMDAR- mediated train-EPSCs to the postsynaptic summation of EPSCs (data not shown). Normalized charge transfer of the train-EPSCs from Tscl+/- mice was 0.093±0.003 in control and 0.055±0.010 in the presence of UBP 141 (n=5 cell pairs, p<0.009 (two-tail t-test)), while in Tsclwt mice normalized charge transfer of the train-EPSCs was unaltered in the presence of UBP141 (data not shown). Thus, abnormal slowing of the NMDAR-mediated current kinetics increases temporal integration within recurrent network in L4 of Tscl+/- mice.

4. The selective antagonists of GluN2C/D subunit-containing NMD A receptors reduce epileptiform activity in Tscl+/- mice

The critical role of L4 neurons in seizure generation suggests that the selective blockade of long-lasting GluN2C/D subunits containing NMDAR-mediated currents may have antiepileptic effects. To test this possibility, we first used microelectrode array extracellular recordings in acute coronal neocortical slices taken from P15 Tscl+/- mice. Spontaneous discharges lasting for up to 10 seconds were recorded in L2/3 and L4. UBP141 (10 μΜ) selectively reduced the amount of long-lasting epileptiform episodes, without altering the number of interictal bursts (< 500 ms) (data not shown).

We next tested the antiepileptic actions of UBP 141 and DQP1105 in vivo by intraperitoneal (IP) injections of the drugs (75 mg/kg and 28 mg/kg respectively) to Tscl+/- mice. These values were the least effective doses identified by testing increasing doses of these compounds. In 3 of 6 mice tested the seizures were completely stopped -40 min after IP injection of UBP141 and in the remaining mice (n=3) there was a seizure free period lasting 109±39 minutes (Fig. 4a,b). IP injection of DQP1 105 stopped seizures on average for 72.6±5.5 minutes in 4 out of 6 mice and seizures were completely stopped in 2 mice.

In contrast, recurrent epileptic discharges persisted up to 7 hours in Tscl+/- mice that did not receive the antagonists or were IP injected with saline (data not shown).

The concentration of UBP 141 used in in vivo experiments (200 μΜ) is far from the range of selectivity for GluN2C/D (data not shown). However, assuming limited predicted blood brain barrier permeability of this compound (log of partition coefficient (ClogP)=1.79; polar surface area=106.71), we expect that ACSF concentration of UBP141 will be within the selectivity range for UBP141 (see Methods and Supplementary table 1). Importantly, however, after UBP141 injection a basal activity remained unaltered, in particular, at γ frequency band known to be enhanced by common NMDAR antagonists (data not shown). On the other hand, the concentration of DQP1105 (50 μΜ) used is selective for GluN2C/D receptor (data not shown).

Therefore, selective antagonists of GluN2C/D containing receptors have in vivo and in vitro antiepileptic actions in Tscl+/- mice.

Collectively our observations on the Tscl animal model suggest that seizures are generated intra-cortically due to an up-regulation of GluN2C receptors in recurrent connections between SSC in the granular layer of neocortex and then propagate to other layers. Interestingly, tactile stimulation of the Tscl+/- mouse back induced prominent seizures (data not shown), indicating that seizure initiation can be triggered by conventional sensory inputs to L4. This strongly reinforces the importance of the hyper- synchronizing effects of L4 and of functional up-regulation of GluN2C subunits of NMDARs.

5. Increased GluN2C expression in post-surgical tissue from epileptic patients with TSC and focal cortical dysplasia

To test whether findings obtained in the animal model can be translated to human patients with TSC, we performed studies in human postsurgical tissue. Quantitative RT-PCR performed in 3 human samples with TSC2 mutation (age at surgery ranged from 8 to 16 months, data not shown) revealed a 20-fold increase of GluN2C mRNA compared to fetal control brains (data not shown, p<0.001) and more than 2.5 -fold increase compared to adult control brains.

Whole-cell patch-clamp recordings in brain slices from the same human TSC specimens showed that sEPSCs recorded from dysplastic neurons in granular and supragranular layers (data not shown) were significantly reduced and curtained by UBP141 (Fig. 5c,d). The amplitude, and total and normalized charges of sEPSC in the presence of UBP141 (10 μΜ) were 0.78±0.04 (p<0.002, t-test, n=7), 0.59±0.08 (p<0.002, t-test, n=7), and 0.68±0.09 (p<0.01, t-test, n=7), respectively. The values of Dw of sEPSC decay in TSC samples were 16.6±2.1 ms without UBP141 and 8.69±0.71 ms in the presence of UBP141 (p<0.0002, t-test, n=7).

Furthermore, in two human tissue samples displaying spontaneous paroxysmal activity UBP141 (10 μΜ) reduced spontaneous spike frequency (to 57.3±0.06% of control) (data not shown). Therefore in conjunction with animal model data functional upregulaton of GluN2C may contribute to epilepsy associated with TSC in human patients.

Genetic polymorphisms and biochemical markers of mTOR activation have been also identified in patients with isolated focal cortical dysplasia (FCD), a common etiology of intractable epilepsy 48-52. GluN2B and GluN2C mRNA levels have been found to be upregulated in dysplastic neurons microdissected from human focal cortical dysplasia specimens obtained during epilepsy surgery. Therefore, we tested whether slow UBP141- sensitive NMDAR-mediated component is present in dysplastic neurons from patients with FCD. Whole-cell patch-clamp recordings were performed in 6 human samples (age at surgery ranged from 1 to 14 years, Supplementary Table 4). As in the TSC case, sEPSCs recorded from dysplastic neurons in granular and supragranular layers were significantly curtained by UBP141 (data not shown). The total and normalized charge of sEPSC in the presence of UBP141 (10 μΜ) were 0.56±0.08 (p<0.005, t-test, n=12) and 0.6±0.04 (p<0.0002, t-test, n=12), respectively. Tw of sEPSC decay were 22.7±2.9 ms in control (n=17) and 9.9±0.7 ms in the presence of UBP (n=13) (p<0.001, two-samples t-test). Amplitudes of sEPSC in the presence of antagonists were not significantly different from control (data not shown).

Thus, although our direct results are designed for studying the mechanisms of epileptogenesis associated with TSCl mutation, they may provide important link for other neurodevelopmental disorders with epilepsy associated with mTOR activation such as FCD.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Acker, T.M. et al. Mechanism for noncompetitive inhibition by novel GluN2C/D N- methyl-D-aspartate receptor subunit-selective modulators. Mol Pharmacol 80, 782-95 (2011).

Ben-Ari Y. Neuro-archaeology: pre-symptomatic architecture and signature of neurological disorders. Trends Neurosci. 2008 Dec;31(12):626-36. Epub 2008 Oct 24.

Blaise Mathias Costa, Bihua Feng, Timur S. Tsintsadze, Richard M. Morley, Mark W. Irvine, Vera Tsintsadze, Natasha A. Lozovaya, David E. Jane, and Daniel T. Monaghan. N- Methyl-D-aspartate (NMDA) Receptor NR2 Subunit Selectivity of a Series of Novel Piperazine-2,3-dicarboxylate Derivatives: Preferential Blockade of Extrasynaptic NMDA Receptors in the Rat Hippocampal CA3-CA1 Synapse. J Pharmacol Exp Ther. 2009 Nov;331(2):618-26. Epub 2009 Aug 14.

Feldmeyer, D. Excitatory neuronal connectivity in the barrel cortex. Front Neuroanat

6, 24 (2012).

Feng B, Tse HW, Skifter DA, Morley R, Jane DE, Monaghan DT. Structure-activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: l-(phenanthrene-2- carbonyl) piperazine-2,3-dicarboxylic acid. Br J Pharmacol. 2004 Feb; 141(3):508-16. Epub 2004 Jan 12.

Feliciano, D. & Bordey, A. [Mouse model of tuberous sclerosis complex]. Med Sci (Paris) 27, 328-30 (201 1).

Hagino Y, Kasai S, Han W, Yamamoto H, Nabeshima T, Mishina M, Ikeda K. Essential role of NMDA receptor channel ε 4 subunit (GluN2D) in the effects of phencyclidine, but not methamphetamine. PLoS One. 2010 Oct 28;5(10):el3722.

Kinarsky L, Feng B, Skifter DA, Morley RM, Sherman S, Jane DE, Monaghan DT. Identification of subunit- and antagonist-specific amino acid residues in the N-Methyl-D- aspartate receptor glutamate-binding pocket. J Pharmacol Exp Ther. 2005 Jun;313(3): 1066- 74. Epub 2005 Mar 2.

Koller Manuel, Stephan Urwyler. Novel N-methyl-d-aspartate receptor antagonists: a review of compounds patented since 2006. Expert Opinion on Therapeutic Patents Dec 2010, Vol. 20, No. 12, Pages 1683-1702: 1683-1702.

Qin, W. et al. Analysis of TSC cortical tubers by deep sequencing of TSC 1 , TSC2 and KRAS demonstrates that small second- hit mutations in these genes are rare events. Brain Pathol 20, 1096-105 (2010).

Thompson C.L., D.L. Drewerya, H.D. Atkinsa, F.A. Stephensonb, P.L. Chazot. Immunohistochemical localization of N-methyl-D-aspartate receptor NRl , NR2A, NR2B and NR2C/D subunits in the adult mammalian cerebellum. Neuroscience Letters 283 (2000) 85- 88.

Claims

CLAIMS:

1. A compound which is an antagonist of NR2C/D receptor or an inhibitor of the NR2C/D receptor expression for use in prevention or treatment of epileptic seizures.

2. A compound according to the claim 1 wherein the epileptic seizures are developmental epilepsies, infantile epilepsies or adult epilepsies.

3. A compound according to the claims 1 or 2 wherein the epileptic seizures are epilepsy associated with tuberous sclerosis complex.

4. A compound according to the claims 1 or 2 wherein the epileptic seizures are epilepsy associated with focal cortical dysplasia.

5. A compound according to the claims 1 or 2 wherein the epileptic seizures are epilepsy associated with mTORpathies.

6. A compound according to claims 1 to 5, wherein said compound is an antagonist of NR2C/D receptor.

7. A compound according to claims 1 to 6 wherein the compound is the UBP141.

8. A compound according to claims 1 to 6 wherein the compound is the DQP1105.

9. A pharmaceutical composition for use in the prevention and treatment of epileptic seizures comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.