ES2561728A1 - Animal non-human model for transtornes of the autista spectrum, anxiety and/or depression (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Non-human animal model for autism spectrum disorders, anxiety and/or depression. The invention discloses a transgenic animal model for disorders of the autistic spectrum, anxiety and/or depression, stably comprising a heterologous nucleotide sequence encoding the gluk4 protein, in which said nucleotide sequence is integrated into its genome. It is expressed in the animal's previous brain. Therefore, the invention relates to a method for generating this model and a method for testing candidate drugs and drugs for the treatment of autism spectrum disorders, anxiety and/or depression. (Machine-translation by Google Translate, not legally binding)

Description

5

10

fifteen

twenty

25

30

35

NON-HUMAN ANIMAL MODEL FOR DISORDERS OF THE AUTISTIC SPECTRUM.

ANXIETY AND / OR DEPRESSION

DESCRIPTION

The present invention relates to a non-human transgenic animal useful as an animal model for autism spectrum disorders, anxiety and / or depression. The non-human transgenic animal of the invention comprises in its genome a stably integrated heterologous nucleotide sequence encoding the GluK4 protein, in which said nucleotide sequence is expressed in the animal's forebrain (forebrain). Therefore, the present invention belongs to the technical field of the treatment of neurological disorders, mainly autism, anxiety or depression.

BACKGROUND OF THE INVENTION

Autism spectrum disorders or ASDs (collectively referred to herein as autism) include a group of serious and enigmatic neurobehavioral disorders that usually manifest in childhood and persist for a lifetime. The ASDs comprise a set of developmental disorders that alter social interaction and communication, cause in the affected feelings and unstable emotions and, in many cases, affect cognitive development. ASDs are also called generalized developmental disorders, since it is not a term that designates a type of developmental disorder, but a term that designates, in a broad sense, diseases that share several characteristic symptoms. In recent years, the prevalence of ASDs in each country has increased, and therefore the social and economic costs associated with ASD have increased and the quality of life of the patient's family has decreased. For this reason, studies on the establishment of the cause of ASD and the development of agents for the treatment of ASD have been increasingly necessary. With respect to the cause of ASD, several risk factors have been studied, and the genetic tendency, viral infection, abnormal metabolism and other environmental factors including parenting methods have typically been recognized as risk factors and causes.

In recent years, studies have been carried out in animal models on the influence of certain genetic factors of ASD, and through genetic studies in

5

10

fifteen

twenty

25

30

35

Several patients with ASD have found several genes that are believed to be closely related to this disorder. Because ASDs show high heritability in comparison to other mental disorders, such studies on the genetic factors of autism are important to broaden the understanding of the biological causes of ASDs and to develop effective therapeutic agents against them.

Animal models can provide a valuable resource for the study of neuropathologies, as well as for the testing of therapeutic compounds and treatments aimed at delaying or stopping the disorder they mimic. Due to the genetic complexity of autism spectrum disorders, strains of mice with a deletion or mutations in autism-related genes have become an essential tool for investigating the underlying molecular and neurological development mechanisms of ASD. The available mouse models for autism can be found in the Simon Foundation Autism Research Initiative (SFARI). Syndromic ASD genes are defined as those genes that predispose to autism in the context of a syndromic disorder. These include, but are not limited to, CNTNAP2, FMR1, MECP2, NF1, PTEN, SHANK3, TSC1 / 2 and UBE3A (Provenzano, G. et al. 2012. Disease Markers 33, 225-239).

During development, the human brain is made up of sensory inputs and cognitive experiences. In this period, the structure and function of neuronal connections, essential for brain performance, can be altered by changes in the molecular configuration of their synapses. In the glutamatergic system, for example, hyper or hypo-activity induced by several factors represents an attractive model for studying the pathophysiology of serious mental disorders. In fact, a chromosomal abnormality has been found that alters a gene that codes for an ionotropic glutamate receptor of the kainate class in individuals with schizophrenia and in individuals with schizophrenia and mental retardation (Pickard, BS et al. 2012. Human Molecular Genetics 21 , 3513-3523). The genes that direct the synoptic function also seem to influence the risk of autism (Levy D. et al. 2011. Neuron 70, 886-897; Gilman, SR et al. 2011. Neuron 70, 898-907) and, interestingly, some of them belong to the family of glutamate receptor subunits of the kainate type, such as GRIK2 and GRIK4 (Griswold, AJ et al. 2012. Human Molecular Genetics 21, 3513 to 3523). Recent studies indicate that de novo germline mutations and numerical copy variants represent the factors of

5

10

fifteen

twenty

25

more significant risk for autism spectrum disorders than previously recognized. This is the case of GRIK4, assigned to cytobanda 11q23.3-q24.1, which was found duplicated de novo in an autism case (Griswold, A.J. et al. 2012 cited ad supra). Currently, there are no representative animal models of ASD from germline mutations and / or copy number variants.

Anxiety disorders are some of the most prevalent psychological disorders. Anxiety is an emotion, connected to the body's response to stressors, while risk factors can be avoidable. Anxiety disorders are classified as anxiety disorders not related to stress or stress-related. Anxiety disorders related to stress include adjustment disorder and acute stress response; Anxiety disorders not related to stress are panic disorder and generalized anxiety disorder. Anxiety can be studied in animal models. There are models of animals where the anxiety trait or anxiety state has been induced in the laboratory. For example, patents US6,353,152 and US6,060,642 represent animal models for anxiety. However, the animal models described in these patents reach a low degree of anxiety and do not mimic acute episodes of anxiety in humans.

In view of the foregoing, there is a need in the state of the art to provide new non-human animal models for autism spectrum disorders, anxiety and / or depression that represent the multiple phenotypes of these disorders in humans.

5

10

fifteen

twenty

25

30

35

The present invention generally relates to a non-human transgenic animal that can be used as a non-human animal model for the study of autism spectrum disorders, anxiety and / or depression. Said transgenic animal is characterized in that it comprises in its genome a heterologous nucleotide sequence encoding the stably integrated GluK4 protein, wherein said nucleotide sequence is expressed in the anterior brain of the transgenic animal. As a consequence, the level of GluK4 protein in the transgenic animal (the level resulting from the sum of the homologous and heterologous GluK4 protein levels) is increased with respect to the level of said protein in the non-transgenic animal.

The mice expressing GluK4 of the present invention were generated by introducing 5 copies of the Myc tag in phase with the start codon of GRIK4 and under the control of the CaMKII promoter. Next, the physiological and behavioral consequences of the overexpression of the GRIK4 gene in the anterior brain of the mouse were evaluated using a battery of electrophysiological and behavioral tests. As can be seen in Example 1, the results obtained showed that the transgenic animal model generated reproduces some endophenotypes observed in autism, mimicking the situation in humans with ASD.

Based on this discovery, the inventors have developed several inventive aspects that are described in detail below.

The non-human transgenic animal of the invention

Therefore, one aspect of the present invention relates to a non-human transgenic animal that comprises stably integrated into its genome a heterologous nucleotide sequence encoding the GluK4 protein, in which said nucleotide sequence is expressed in the brain. anterior of the animal (hereinafter "non-human animal of the invention").

The term "stably integrated into its genome" refers to a nucleotide sequence that is well integrated into a chromosome in a cell.

5

10

fifteen

twenty

25

30

35

or in an animal, for example, in an endogenous chromosome or as part of an artificial chromosome, or is present in an extrachromosomal form that is not diluted or lost during cell divisions during the life of the cell. For example, a viral vector that does not integrate into the genome of a host cell, such as an adenoviral vector, is known as stably integrated into the genome of a cell, if the cell is a cell that does not divide, such as a post mitotic neuron. Therefore, in the context of the present invention, the heterologous nucleotide sequence encoding the GluK4 protein is integrated into a chromosome of the non-human animal of the invention and is not diluted or lost during the life of the cell. The descendants of these transfected cells, therefore, also express the new gene, resulting in a stably transfected cell line.

The term "heterologous" refers to a gene or part of a gene in a host organism that is derived from a different species than the host organism. Therefore, in the context of the present invention, the nucleotide sequence encoding the GluK4 protein is an exogenous nucleotide sequence with respect to the non-human transgenic animal. Said gene that derives from a different species of the host / animal cell can also be called a "transgene."

The GluK4 protein is a subtype of kainate receptor belonging to the family of ligand activated ion channels that is encoded by the GRIK4 gene. A nucleotide sequence encoding any GluK4 protein can be used in the context of the present invention. Examples of GluK4 proteins include, but are not limited to, rat GluK4 protein (Rattus norvegicus), human GluK4 protein (Homo sapiens), mouse GluK4 protein (Mus musculus), mole rat GluK4 protein (Nannospalax) galili), horse GluK4 protein (Equus przewalskii), rabbit GluK4 protein (Oryctolagus cuniculus), Chinese Hamster GluK4 protein (Cricetulus griseus) and Common chimpanzee GluK4 protein (Pan troglodytes). In a particular embodiment, the GluK4 protein is the rat GluK4 protein.

The amino acid sequences of these proteins are available in public databases and can be easily recovered by the person skilled in the art. However, examples of amino acid sequences of GluK4 proteins include, but are not limited to, Rattus norvegicus GluK4 protein (SEQ ID NO: 1, NCBI accession number NP_036704 version NP_036704.1 GI: 10242377),

5

10

fifteen

twenty

25

30

35

Homo sapiens GluK4 protein (SEQ ID NO: 3, NCBI accession number AAI46653.1 version AAI46653.1 Gl: 148921519), Mus musculus GluK4 protein (SEQ ID NO: 4, NCBI accession number Q8BMF5, version Q8BMF5. 2 Gl: 341940775), Nannospalax galili GluK4 protein (SEQ ID NO: 5, NCBI accession number XP_008824232, Version XP_008824232.1 Gl: 674105958), Equus przewalskii GluK4 protein (SEQ ID NO: 6, NCBI number access XP_008511947, version XP_008511947.1 Gl: 664709971), the GluK4 protein from Oryctolagus cuniculus (SEQ ID NO: 7, NCBI accession number XP_008258709.1, version XP_008258709.1 Gl: 655601372), the GLUK4 protein from Cricetulus gríse SE (GríseQ gríse gríse) ID NO: 8, NCBI accession number XP_007631296, version XP_007631296.1 Gl: 625279300), and Pan troglodytes GluK4 protein (SEQ ID NO: 9, NCBI accession number Q5IS46, Gl version Q5IS46.1: 61213662). In another particular embodiment, the GluK4 protein comprises the amino acid sequence of SEQ ID NO: 1. It should be noted that the invention is not limited to these specific sequences and that additional GluK4 protein sequences are known in the state of the art.

In view of the foregoing, the person skilled in the art understands that the GluK4 protein to be used in the present invention can be derived from several sources, said proteins being functionally equivalent variants of each. Thus, in the context of the present invention, functionally equivalent variants of SEQ ID NO: 1 are also contemplated. The term "functionally equivalent variant of SEQ ID NO: 1" refers to a protein (or polypeptide) comprising one or more more alterations, such as substitutions, insertions, deletions and / or truncations of one or more specific amino acid residues at one or more specific positions in the protein, and having the same function as SEQ ID NO: 1, that is, belongs to the family of glutamate receptors, mediating excitatory synoptic transmission. An assay to assess whether the protein is a functionally equivalent variant of SEQ ID NO: 1 is, for example, the assay described in the Examples of the patent application and which comprises the measurement of KAR mediated by postsynaptic currents. excitatory (EPSCkar) in the non-human transgenic animal comprising the variant. It would also be possible to test the functionality of the protein by performing an assay such as that described in Figure 1b, where the exogenous nucleotide sequence has been expressed in HEK293 cells together with a plasmid encoding GluK2 subunits, where the current only it could be induced by the ATPA agonist provided that the product of said nucleotide sequence heteromerice with GluK2 subunits to form functional receptors.

5

10

fifteen

twenty

25

30

35

Therefore, the GluK4 protein variants may be (i) one in which one or more of the amino acid residues are substituted with a conserved or unconserved amino acid residue (preferably a conserved amino acid residue) and such a residue of Substituted amino acid may or may not be one encoded by the genetic code, (ii) one in which there is one or more modified amino acid residues, for example, residues that are modified by the binding of substituent groups, (iii) one in the that the protein is a processing variant (splice-variant) of the proteins of the present and / or (iv) fragments of the invention proteins. Fragments include proteins generated through proteolytic cleavage (proteolysis including multi-site) of an original sequence. Variants are considered within the scope of those skilled in the art from the teaching herein.

Therefore, in a particular embodiment, the GluK4 protein comprises an amino acid sequence with a sequence identity of at least 95, 96, 97, 98 or 99% at SEQ ID NO: 1. In another particular embodiment, the protein GluK4 comprises the amino acid sequence of SEQ ID NO: 1.

In the context of the present invention, the term "sequence identity" refers to the homology between two amino acid sequences. The amino acid sequences are said to be homologous by exhibiting a certain level of similarity. If two homologous sequences are closely related or further away, it is indicated by "percent identity" or "percent similarity", which is high or low, respectively. As is known in the art, the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of a protein to a sequence of a second protein. The degree of identity between two proteins is determined using computer algorithms and methods that are widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined by the BLASTP algorithm [BLASTM Annual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

The nucleotide sequences encoding the GluK4 proteins mentioned above can be obtained from public databases, such as the National Center for Biotechnology Information (NCBI). However, in one embodiment

5

10

fifteen

twenty

25

30

35

In particular, the nucleotide sequence encoding the GluK4 protein comprises the nucleotide sequence SEQ ID NO. 2 (Rattus norvegicus, NCBI accession number NM_012572, version NM_012572.1 Gl: 10242376).

One of the characteristics of the non-human animal of the invention is that the nucleotide sequence encoding the GluK4 protein is expressed in the animal's anterior brain.

The term "anterior brain," also called the forebrain, refers to the anterior part of the three major brain divisions of developing vertebrates. The anterior brain is the largest part of the brain, most of it is formed by the cerebral cortex. Other important structures found in the anterior brain include the thalamus, the hypothalamus and the limbic system (a collection of structures within the anterior brain, including the amygdala and the hippocampus).

Therefore, in a particular embodiment, the nucleotide sequence is expressed in the hippocampus and / or in the neocortex.

The term "hippocampus" refers to the part of the brain that is involved in the formation, organization and storage of memory. It is found under the cerebral cortex; and in primates it is found in the medial temporal lobe, below the cortical surface. It contains two intertwined main parts: the horn of Amun and the dentate gyrus.

The term "neocortex" or "neocortex" refers to most of the cerebral cortex, which encompasses the two cerebral hemispheres, formed by six layers, labeled from the outside, I to VI.

Any method known in the state of the art for creating transgenic animals can be used to obtain the non-human transgenic animal of the invention. These will be disclosed in detail later in other aspects of the invention.

As the skilled person will understand, the nucleotide sequence must be linked to an expression regulatory sequence that controls the transcription of said

5

10

fifteen

twenty

25

30

35

nucleotide sequence Therefore, in another particular embodiment, the heterologous nucleotide sequence encoding the GluK4 protein is operably linked to an expression regulatory sequence. An expression regulatory sequence is a segment of a nucleic acid molecule that refers to a promoter sequence or an upstream sequence (such as a promoter), in which the activation thereof regulates (increasing or decreasing ) the transcription of a nucleic acid sequence operatively linked thereto. As used in this description, the term "operably linked" means that the polynucleotide is within the correct reading pattern for expression under the control of said regulatory sequences. The regulatory sequences useful for the present invention may be nuclear sequences that promote or, alternatively, enhancer sequences and / or other sequences that regulate the increase in polynucleotide expression. In another more particular embodiment, the expression regulatory sequence is a promoter and / or an enhancer.

The promoter can be constitutive or inducible. If the constant expression of the polynucleotide is desired, then a constitutive promoter is used. Examples of well-known constitutive promoters include the immediate early cytomegalovirus (CMV) promoter, the Rous sarcoma virus promoter, the CAGS promoter, a C-ubiquitin promoter, and the like. A large number of other examples of constitutive promoters are well known in the art and can be used in the application of the invention. If controlled expression of the polynucleotide is desired, then an inducible promoter should be used. The level of expression can often be controlled by varying the concentration of the inducer so that the inducible promoter is stimulated more strongly or weakly and, consequently, the concentration of the transcribed product of the polynucleotide is affected. Examples of well-known inducible promoters are: an androgen or estrogen sensitive promoter, a doxycycline sensitive promoter, a metallothionein promoter, or an ecdysone response promoter. Other examples are well known in the art and can be used to implement the invention. In addition, a specific expression of the nucleotide sequence of interest may be desired. In this case, a cell type specific promoter or a tissue specific promoter can be used in the present invention. For example, the promoter can be specific to neuronal cells, specific to the brain, specific to neurons, or specific to glial cells, for example, the tau promoter, the CaMK promoter, the Nestine promoter, the GAFP promoter, the Tubulin III promoter, or other known promoter

5

10

fifteen

twenty

25

30

35

by those skilled in the art for being specifically active in one of these tissue cells. Therefore, in a particular embodiment, the promoter is a specific promoter of the Central Nervous System, such as the Neuron Specific Enolase (NSE) promoter. In a particular embodiment, the promoter is the CaMKII alpha promoter or the GRIK4 gene promoter

As mentioned above, the non-human transgenic animal of the invention is characterized in that it comprises stably integrated into its genome a heterologous nucleotide sequence encoding the GluK4 protein, in which said nucleotide sequence is expressed in the animal's anterior brain. transgenic As a consequence, the level of GluK4 protein in the transgenic animal (the level resulting from the sum of homologous and heterologous GluK4 protein levels) is increased with respect to the level of said protein in the non-transgenic animal. The presence of an increase in GluK4 protein levels in the animal may be due to the presence in the genome of more than one copy of the heterologous nucleotide sequence encoding the GluK4 protein. Therefore, in another particular embodiment, the non-human transgenic animal of the invention comprises at least 2, 3 or 4 copies of the heterologous nucleotide sequence encoding the GluK4 protein.

Any non-human animal can be used to express the nucleotide sequence encoding the GluK4 protein in its genome, obtaining the non-human transgenic animal of the invention. Examples of non-human animals include, but are not limited to, mammals, preferably, rodents (mouse, rat, guinea pig, rat-mole, etc.), non-human primates, such as monkeys or chimpanzees. Therefore, in another particular embodiment, the non-human transgenic animal of the invention is a mammal, preferably selected from the group consisting of monkey, pig, rat and mouse.

The transgenic non-human animal of the invention can have any genetic background of those known in the state of the art by one skilled in the art, that is, said non-human transgenic animal can come from a wild-type animal (WT) or from a non-human animal with a genetic background incorporating any molecular marker that is directly or indirectly related to autism, anxiety or depression.

Uses of the non-human transgenic animal of the invention

5

10

fifteen

twenty

25

30

35

As explained above, the non-human transgenic animal is characterized in that it comprises a heterologous nucleotide sequence protein that stably encodes GluK4 inserted into its genome, causing an increase in GluK4 protein levels. Surprisingly, this causes the behavior of transgenic animals to mimic the human symptoms of ASD, depression and / or anxiety. Consequently, the non-human transgenic animal of the invention is useful as a non-human animal model of said diseases.

In another aspect, the invention relates to the use of the non-human transgenic animal of the invention as an animal model for autism spectrum disorders, anxiety and / or depression.

The invention is directed to the use of a non-human transgenic animal of the invention for the detection, discovery, identification, evaluation and validation of compounds potentially useful for the treatment and / or prevention of ASD, depression and / or anxiety.

Therefore, in another aspect, the invention relates to a method for the detection, discovery, identification, evaluation and / or validation of a compound for the treatment of ASD, anxiety and / or depression, hereinafter method of the invention, which includes:

(a) administering a candidate compound to a non-human transgenic animal of the invention and

(b) determine the effect of the candidate compound on the behavior of said non-human transgenic animal, or alternatively, the determination of the level of expression of the GluK4 protein.

As used herein, the terms "autism" and "autism spectrum disorders" (ASDs) are used interchangeably to describe in general three of the five developmental disorders described in Diagnostic and Statistical Manual IV (DSM IV-TR): autistic disorder, Asperger's disorder and generalized developmental disorder not specified (American Psychiatric Association, 2000). The clinical characteristics of autism include alterations in three categories of interactions of social reciprocal behavior, verbal and nonverbal communication, and activities of their age and interests.

5

10

fifteen

twenty

25

30

As used here, the term "anxiety" refers to a fear of an undefined object, that is, an offensive and agonizing emotional reaction that we have in a threatening and dangerous situation that can result in negative results. The term "anxiety disorders" refers to a mental disorder in which anxiety arises without reason and a level of anxiety is too high. That is, this term refers to a case in which excessive psychological distress arises due to morbid anxiety or severe difficulty and that occurs in adaptation to real life. Examples of anxiety disorders include, but are not limited to, a panic disorder, phobia, an obsessive-compulsive disorder, a post-traumatic stress disorder, acute stress disorder, generalized anxiety disorder, and a separation anxiety disorder.

A "panic disorder" is a symptom of extreme anxiety in which fear arises suddenly and for no reason, followed by suffocation or strong heartbeat. That is, panic disorder is a disorder that has several events of panic attack and that can occur due to fatigue, excitement, sexual behaviors, or emotional impacts. However, in general, panic disorder cannot be predicted and occurs suddenly.

"Phobia" refers to an anxiety disorder characterized by avoiding a particular situation or subject due to anxiety and fear of the particular situation or serious issue. 1) Specific phobia is a disorder characterized by recurrent irrational fear and avoidance behaviors of a particular issue or situation. 2) Social phobia is a disorder that is characterized by repeatedly showing evasive behaviors for fear of a social situation where interaction between people occurs. 3) Agoraphobia is a disorder that is characterized by showing on several occasions the fear of a particular place or situation.

"Obsessive-compulsive disorder" is an anxiety disorder characterized by recurring unwanted thoughts, that is, obsessions and behaviors.

"PTSD" is an anxiety disorder characterized by persistent anxiety in response to a terrifying event.

5

10

fifteen

twenty

25

30

35

"Acute stress disorder" is an anxiety disorder that shows symptoms similar to those of posttraumatic stress disorder and is characterized by showing dissociative symptoms in response to a traumatic event.

"Generalized anxiety disorder" is an anxiety disorder characterized by chronic anxiety and excessive worry about various situations.

"Separation anxiety disorder" is a condition in which an individual experiences excessive anxiety regarding the separation of people to whom the individual has a strong emotional attachment. If separation anxiety exceeds a normal range and interferes with a normal social life, this can be defined as a morbid state. In this regard, separation anxiety disorder refers to a case in which separation anxiety is excessive and therefore interferes with normal activities.

As used herein, the term "depression" refers to a disorder of the brain that comprises the feelings and symptoms in an individual that interfere with his ability to work, sleep, study, eat and enjoy life. Examples of depressive disorders include, but are not limited to, persistent major depressive disorder [such as psychotic depression, postpartum depression, seasonal affective disorder (SAD)] and bipolar disorder.

The term "major depression" refers to severe symptoms that interfere with the ability to work, sleep, study, eat and enjoy life. An episode can occur only once in a person's life, but more often, a person has several episodes.

The term "persistent depressive disorder" refers to a depressive mood that lasts at least 2 years. Some forms of depression are slightly different, or may develop under unique circumstances. These include, but are not limited to:

• Psychotic depression, which occurs when a person suffers from severe depression plus some form of psychosis, such as having disturbing false beliefs or a break with reality (delusions) or seeing or hearing disturbing things that others cannot hear or see (hallucinations) ).

5

10

fifteen

twenty

25

30

35

• Postpartum depression, which is much more serious than the "baby blues" many women experience after giving birth, when hormonal and physical changes and the new responsibility of caring for a newborn can be overwhelming. It is estimated that 10 to 15 percent of women experience postpartum depression after giving birth.

• Seasonal affective disorder (SAD), which is characterized by the appearance of depression during the winter months, when there is less natural sunlight. Depression usually rises during spring and summer. SAD can be treated effectively with light therapy, but almost half of people with SAD do not improve with light therapy alone. Antidepressant medications and psychotherapy can reduce the symptoms of SAD, either alone or in combination with light therapy.

The term "bipolar disorder", also called manic-depressive illness, refers to mood swings, cycling in the individual from the maximum extremes (for example, mania) to extreme casualties (for example, depression).

In a first step, the method of the invention comprises administering a candidate compound to a non-human transgenic animal of the invention.

The candidate compounds or under test may be compounds of any nature, for example, a chemical, biological, microbiological, etc. compounds isolated or mixed with one or more different compounds, and include compounds with a known or unknown composition and structure, pharmaceuticals with any known therapeutic application, biological products, microbiological products, etc., for example, organic or inorganic chemical compounds, peptides, proteins, nucleic acids, extracts, etc.

In a second step, the method of the invention comprises determining the effect of the candidate compound on the behavior of said non-human transgenic animal.

In the non-human transgenic animal of the invention some endophenotypes seen in autism, anxiety and / or depression are reproduced, for example, impaired social behavior, a deficit in their social interaction, less activity than in non-transgenic animals, the lack of motivation (increase of sedentary periods), anhedonia, repetitive behavior, defective communication (for

5

10

fifteen

twenty

25

30

35

example, vocalization), etc .; if after administering the candidate compound to the non-human transgenic animal of the invention, the animal changes one or more of the aforementioned endophenotypes to behaviors with more social interaction, increased activity, less sedentary periods, etc .; then it can be concluded that the candidate compound is useful for treating autism spectrum disorders, anxiety and / or depression.

The term "treatment" or "treat" refers to improving, attenuating, decreasing or eliminating one or more of the symptoms of autism spectrum disorders, anxiety and / or depression. Therefore, in the context of the present invention, a compound is considered to be useful for treating autism spectrum disorders, anxiety and / or depression when one or more of the symptoms associated with said disorders is improved, attenuated, diminished or eliminated ( due to the administration to the subject of said composition).

The change in the behavior of the non-human transgenic animal of the invention after administering the test compound can be evaluated by using a battery of behavioral tests. Such tests are widely known in the state of the art and in routine practice for the person skilled in the art. Any of them can be used in the context of the present invention. Behavioral tests include, but are not limited to, the three chamber social interaction test, forced swimming test, cross-raised labyrinth, open field testing and the light-dark box.

Alternatively, the second step of the method of the invention may comprise determining the level of expression of the GluK4 protein. As explained above, the non-human transgenic animal of the invention is characterized in that it comprises stably integrated into its genome a heterologous nucleotide sequence encoding the GluK4 protein and, as a consequence, the level of GluK4 protein in the transgenic animal (the level resulting from the sum of homologous and heterologous GluK4 protein levels) is increased with respect to the level of said protein in the non-transgenic animal. In the context of the present invention, the term "homologous" is equivalent to the term "endogenous" and both refer to the GluK4 protein expressed naturally by the animal.

5

10

fifteen

twenty

25

30

35

Therefore, if after administering the candidate compound to the non-human transgenic animal of the invention, the level of GluK4 protein in the animal (the level resulting from the sum of homologous and heterologous GluK4 protein levels) is less than the level of protein in GluK4 the equivalent non-transgenic animal, is indicative that the candidate compound is useful for treating autism spectrum disorders, anxiety and / or depression.

The methods for determining protein levels in a biological sample are widely known in the state of the art and are routine practice for an expert.

In another aspect, the invention relates to the use of the non-human transgenic animal of the invention in the screening, discovery, identification, evaluation and / or validation of a compound or therapy to treat autism spectrum disorders, anxiety and / or depression.

On the other hand, the non-human transgenic animal of the invention, for example, a mouse, can also be used by crossing with other non-human animals of the same species to obtain non-human animals having the characteristic genotype and phenotype. of their parents. Examples of non-human animals that can cross the non-human animal of the invention include: non-human animals knock-out for the GRIK4 gene, or other genes directly or indirectly related to ASDs, anxiety and / or depression; non-human animals expressing allelic variations, polymorphisms or mutations in genes involved in ASDs, anxiety and / or depression, or other non-human animals that can be used as animal models of ASDs, anxiety and / or depression.

Therefore, in the context of the present invention, the progeny, offspring or descendants of the non-human transgenic animal of the invention are also contemplated.

Methods for producing the non-human transgenic animal of the invention

In another aspect, the invention relates to a method of producing a non-human animal model of autism comprising the expression in the anterior brain of a non-human animal a heterologous nucleotide sequence encoding the

5

10

fifteen

twenty

25

30

35

GluK4 protein, in which the heterologous nucleotide sequence is stably integrated into the genome of the non-human animal.

Methods for expressing genes in non-human animals are widely known to the person skilled in the art. For example, the gene of interest (in the present invention the nucleotide sequence encoding the GluK4 protein or transgene) can be introduced into the non-human animal by using a vector comprising the gene of interest operably linked to a regulatory sequence. , which increases its expression.

In a particular embodiment, the gene of interest encodes the GluK4 protein comprising the amino acid sequence with a sequence identity of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% with SEQ ID NO: 1. In another particular embodiment, the gene of interest encoding the GluK4 protein comprises the nucleotide sequence SEQ ID NO: 2 In another particular embodiment, the GluK4 protein is the rat GluK4 protein.

In another particular embodiment, the regulatory sequence is a promoter and / or an enhancer, preferably, the promoter is a specific promoter of the Central Nervous System, more preferably, the promoters the CaMKII alpha promoter or the GRIK4 gene promoter. In another particular embodiment, the expression comprises the introduction of at least 2, 3 or 4 copies of the heterologous nucleotide sequence encoding the GluK4 protein in the genome of the non-human animal. Therefore, the vector comprises 2, 3 or 4 copies of the transgene. All these particular embodiments have been described in the preceding paragraphs.

The vector can then be introduced into the desired host cell by methods known in the art, for example, transfection, transformation, transduction, electroporation, infection, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, liposomes, LIPOFECTIN ( TM) (Bethesda Research Laboratories, Gaithersburg, MD.), Fusion of lysosomes, synthetic cationic lipids, use of a gene gun or DNA transport vector, such that the transgene is transmitted to the offspring of the line. Particularly preferred embodiments of the invention encompass the methods of introducing the vector containing the transgene by pronuclear injection of a transgenic construct into the mouse nucleus of an embryo and infection with a viral vector.

5

10

fifteen

twenty

25

30

35

which comprises the transgene. In preferred embodiments, a vector containing the transgene is introduced into any nucleic genetic material that ultimately forms part of the zygote nucleus of the animal that will become transgenic, including the zygote nucleus. In one embodiment, the transgene can be introduced into the nucleus of a primordial germ cell that is diploid, for example, a spermatogenesis or an oogonium.

When stable transfection develops, researchers use selection markers to distinguish transient from stable transfections. Co-expression of a marker with the gene of interest allows researchers to identify and select cells that have the new gene integrated into their genome while also being selected against transiently transfected cells. The efficiency of integration depends on the transfection technique and the vector used. In order to identify and select integrating cells, a gene encoding a selectable marker (for example, for antibiotic resistance) is generally introduced into host cells along with the sequence of the gene of interest, for example, the sequence of the gene of interest Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells will die). Such methods are particularly useful in methods that involve homologous recombination in mammalian cells (eg, in murine embryonic stem cells) before the introduction of recombinant cells in mouse embryos to generate chimeras.

A large number of selection systems can be used to select transformed host cells. In particular, the vector may contain certain detectable or selectable markers. Other selection methods include, but are not limited to selecting for another marker such as: herpes simplex thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase virus genes that can be used in tk, hgprt or aprt cells, respectively. . Also, antimetabolite resistance can be used as a selection base for the following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to

5

10

fifteen

twenty

25

30

35

G-418 aminoglycoside; and hygro, which confers resistance to hygromycin (Santerre et al, 1984, Gene 30: 147).

The transgenic non-human animals of the invention can be obtained by any method of transgenesis known to the person skilled in the art comprising introducing the vector described above into the genome of the cell, such as microinjection, electroporation, particle bombardment, cell transformation followed by cloning (nuclei of successfully transformed cells are transferred to enucleated ovules and implanted in recipient females), gamete transformation (introducing genes into oocytes or spermatocytes and the use of transformed gametes for fertilization, generating a complete animal ) and / or intracytoplasmic sperm microinjection (ICSI).

In a particular embodiment of the method, the nucleotide sequence is expressed in the hippocampus and / or in the neocortex.

In another particular embodiment, the non-human animal is a mammal, preferably selected from the group consisting of monkey, pig, rat and mouse.

Once the transgenic mice have been generated they can be raised and maintained using methods well known in the art. By way of example, mice can be housed in a controlled environment facility maintained in a cycle of 10 hours of darkness and 14 hours of light, or with another appropriate light cycle. Mice can mate when they are sexually mature (6 to 8 weeks old). In certain embodiments, the transgenic founders mate with an unmodified animal (ie, an animal that does not have cells containing the transgene), for example, to C57BL / 6 mice (Jackson Laboratories).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by an ordinary expert in the art to which this invention pertains.

All compositions and methods described and claimed herein can be performed and executed without undue experimentation in the light of the present description. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art.

5

10

fifteen

twenty

25

30

35

technique that variations can be applied to the compositions and methods and in the steps or in the sequence of the steps of the method described herein without departing from the scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein, while the same or similar results are achieved.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Generation of mice with over-expression of GluK4 in the anterior brain, a, scheme showing the disposition of the GRIK4 transgene. b, electrophysiological responses induced by glutamate (1 mM) and ATPA (0.1 mM) in HEK293 cells expressing the construct plus GluK2 subunits. The response to ATPA reveals the formation of functional heteromeric GluK2 / K4 receptors. The vertical calibration bars are 100 pA (upper response) and 20 pA (lower part). The horizontal bar is 200 ms. c, in situ hybridization in normal mice (GluK4 + / +) and mice that overexpress GluK4. d, the detection of GluK4 transcribed from the transgene by the anti-myc antibody. e, normal glycolytic processing of exogenous GluK4 protein in GluK4over mice.

Figure 2A-2G GluK4over mice show greater expression of the GluK4 protein and have deficits in social interaction. Figure 2a, Western blot quantification (upper part) of the endogenous protein (GluK4) and the derived transgenic protein (myc-GluK4) in the hippocampus (HPC) and the neocortex (Ncx) of both transgenic mice (Tg) and their littermates who do not carry the transgene (WT). The histograms at the bottom represent the mean ± SEM of measurements of 4 (Tg) and 3 (WT) independent samples performed in duplicate. Figure 2b, the expression levels of other kainate receptor subunits (GluK5 and GluK2 / 3) and AMPA receptor subunits (GluA2 / 3) in the GluK4over transgenic mice and their wild-type litter partners in the cerebral cortex and the hippocampus The histograms at the bottom reflect the mean ± SEM of measurements in 4 (Tg) and 3 (WT) animals performed in duplicate (* p <0.05, Student's t-test). Figures 2c, 2d and 2e, the schematic representation of the three-chamber device is shown at the top with representative examples superimposed of the paths made by GluK4over mice and normal mice in the social interaction test. The histograms at the bottom are

5

10

fifteen

twenty

25

30

35

data quantifications from 11 mice of each genotype. GluK4over mice discriminate well between an inanimate object and a congener (Figure 2c), but contrary to what happens in normal mice (Figure 2d), GluK4over mice spent a similar time in the areas occupied by the new mouse and the family, respectively, indicating alteration of social interaction (Figure 2e). GluK4over mice remained longer in the chamber occupied by family animals, but away from them (circle in Figure 2e) (n = 11 mice of each genotype, *** p <0.001, Student's t-test). Figure 2f, GluK4over mice showed anhedonia: their preference for water with sucrose was lower than that of normal mice (n = 22, p <0.001, Student's t-test). Figure 2g, in the forced swimming test, GluK4over mice remained immobile for longer than normal mice, which is compatible with a depressive state. This behavior was reversed in GluK4-deficient mice (n = 9 mice of each genotype, p <0.001, Student's t-test).

Figure 3. Social interaction test in GluK4over mice. a, representative paths of a mouse that overexpresses GluK4 in the three chamber apparatus. The animal explores both cameras, including where a new mouse (red) and a familiar mouse (gray) are located. The scan ends with the mouse resting in the chamber occupied by the familiar mouse but away from it (arrow), b, quantification (n = 11 mice of each genotype) of the time spent by normal mice and those that overexpress GluK4 in each chamber excluding direct interaction with peers. * p <0.05; ** p <0.01, Student's t-test.

Figure 4. Mice that overexpress GluK4 show severe signs of anxiety, ac, representative paths (above) of normal and GluK4over mice during exploration of a raised cross maze (a) (thicker lines denote closed arms), a open sand (b) and the light-dark box (c). The shaded area in b denotes the area not considered as the center of the stage, which appears as a clear frame. The histograms in the lower part are quantifications (mean ± SEM) of several parameters of each behavior test from 9 (a, b) and 11 animals (c) of each phenotype, p <0.001, Student's t-test.

Figure 5. a, mice that overexpress GluK4 spent more time inactive than their normal littermates (n = 9 mice of each genotype, p <0.001,

5

10

fifteen

twenty

25

30

35

Student t test). b, the transgenic mice and their control litter brothers perform the rotarod test equally well over the days, and show no indication of locomotor impairment. The points are mean ± SEM of 10 experiments in each case.

Figure 6. The synoptic transfer through the trisynaptic circuit of the hippocampus is altered in GluK4over mice. The box in the middle of the figure shows the arrangement of the recording and stimulation electrodes in the in vivo hippocampus of normal animals and GluK4over. a, stimulus-response curve for fEPSP recorded by the electrodes in the molecular stratum of the dentate gyrus (DG) in normal (gray) mice that overexpress GluK4 (black), b, the stimulus-response curves for the recorded population spike just below the granular cell layer of the DG. The same color code is applied, c, Stimulus-response curve for the fEPSP evoked trisinaptically in the CA1 radiated stratum, where the path of the Schaffer collaterals ends. The curves are significantly different (two-way ANOVA, p = 0.002). The inserts in a-c are illustrative examples of averaged responses (n = 9 for GluK4 + / + and 8 for GluK4over). d, The input-output curve was constructed taking into account the amplitudes of the population spike registered in the DG as the input and the amplitude of the trisynaptic EPSP of CA1 as the output of the circuit. The different slopes in the regression line denote a change in the gain of the circuit.

Figure 7. The synoptic transmission of the mossy fibers to the pyramidal cells of CA3 shows an increase in gain in mice that overexpress GluK4. A, hippocampus scheme with recording (Rec) and stimulation (STIM) electrodes (MF, mossy fibers; DG, dentate gyrus). B-c, KAR-mediated spontaneous EPSCs in CA3 pyramidal cells recorded at -60 mV aligned and superimposed membrane potential (gray lines). The resulting average of all responses is presented in a thick line. The mean values of the amplitude (b, right) and deactivation times (c) of these responses are shown for each genotype (n = 28 and 29 neurons from 3 GluK4over and GluK4 + / + mice, respectively, p <0.001, Student t test). d, mPAPSs mediated by the AMPA receptor (Vm = -60 mV) in each genotype (above) and quantification of their frequency and amplitude (bottom) from 22 neurons (2 mice) and 20 neurons (2 mice) of GluK4over and GluK4 + / + mice, respectively; (p <0.001, Student's t-test). e, EPSCs produced by mossy fibers in CA3 neurons (above) and

5

10

fifteen

twenty

25

30

35

its quantification (below) for each genotype (10 neurons of 5 GluK4over mice and 13 neurons from 5 GluK4 + / + mice, p <0.001, Student's t-test). f, pulse pair facilitation of synoptic responses of CA3 at various intervals for each genotype. The points are the mediaiSEM of 22 cells (12 GluK40Ve 'mice) and 32 cells (11 GluK4 + / + mice) (two-way ANOVA and post-hoc Bonferroni test, *** p <0.001; ** p <0, 01, * p <0.05).

Figure 8. Effect of fluoxetine and thianeptin in the GluK4over depression model. Fluoxetine (a serotonin reuptake inhibitor, SSRIs) (10 mg / kg) and thianeptin (8 mg / kg) were injected i.p. for 30 days and then the animals underwent forced swimming tests. The action of these treatments on depression was evaluated by noting the immobility time. As can be seen, thianeptin but not fluoxetine suppresses the signs of depression in GluK4over mice. Number of animals evaluated: 7 WT and 6 GluK4over saline solution; 5 WT and 8 GluK4over for fluoxetine; 9 WT and 10 GluK4over for thianeptin. The values are the mediaiSEM. ANOVA test ** p <0.01, *** p <0.001.

Figure 9. Effect of fluoxetine and thianeptin in the GluK4over ASD model. Fluoxetine (10 mg / kg) and thianeptin (8 mg / kg) were injected i.p. the mice for 30 days after which they were presented with a solution of 3% sucrose in drinking water. The action of these treatments on anhedonia was evaluated by noting the consumption of sucrose in GluK4over mice and their siblings (GluK4 + / +) as controls. As you can see, thianeptin but not fluoxetine normalizes anhedonia in GluK4over mice. Number of animals evaluated: 7 WT and 6 GluK4over saline solution; 5 WT and 8 GluK4over for fluoxetine; 9 WT and 10 GluK4over for thianeptin. The values are the mediaiSEM. ANOVA test * p <0.05.

Figure 10. Effect of thianeptin in the GluK4over anxiety model. Thianeptin (8 mg / kg) was injected intraperitoneally to mice for 30 days and then underwent the elevated labyrinth test (A) or the open field test (B). The action of this treatment on anxiety was assessed by writing the entries in the open arms (A) or the time spent in the center of the arena (B). As can be seen, thianeptin has no action on these two parameters, but it normalizes locomotor activity in GluK4over mice. Number of animals evaluated: 7 WT and 6 GluK4over saline solution; 9 WT and 10 GluK4over for thianeptin. The values are the mediaiSEM. ANOVA test ** p <0.01.

Figure 11. Effect of fluoxetine on the GluK4over anxiety model. Fluoxetine (10 mg / kg) was injected into the mice intraperitoneally for 30 days and then subjected to the open field test. The treatment action on anxiety was evaluated by recording the time spent by the mice in the center of the field. The serotonin reuptake inhibitor, fluoxetine, has no action on the degree of anxiety in GluK4over mice. On the contrary, this antidepressant induces anxiety in normal mice and increases the signs of anxiety in GluK4over mice. Fluoxetine also did not improve locomotion. Number of animals evaluated: 7 WT and 10 6 GluK4over saline solution; 5 WT and 8 GluK4over for fluoxetine. The values are the

mean ± SEM. ANOVA test * p <0.05, ** p <0.01.

5

10

fifteen

twenty

25

30

35

Example 1

Non-human animal model for autism spectrum disorders, anxiety v / o

depression

MATERIAL AND METHODS

The mice that overexpress GluK4 (GluK4over mice) used in this study were generated by the introduction of 5 Myc labels in pattern with the start codon GRIK4 under the control of the CaMKII promoter. Gene expression was evaluated by in situ hybridization, protein levels by immunocytochemistry and in Western blotting with anti-myc mouse and anti-GluK4 rabbit antibodies. Hippocampal and neocortex tissue samples from adult mice were used, and antibodies against GluK2 / 3, GluK5 and GluA2 / 3 were additionally used with the Western blot. The GluK4over mice were evaluated in a series of behavioral tests, including paradigms of social interaction, forced swimming tests for depression, the elevated cross labyrinth, open field stage and the dark light box for anxiety. Neural network activity was evaluated in the hippocampus in vivo using a type of Michigan electrode array to electrophysiologically record the field potentials of the subfields in the CA1 and dentate gyrus areas. The electric shocks applied to the perforating path activate the trisynaptic hippocampus circuit and serve to evaluate the input-output curves. The synaptic transmission of the mossy fibers to CA3 was studied in more detail by patch clamp recordings in hippocampal slices (300 pm) from P19-21 mice. Synaptic transmission mediated by KAR and AMPA receptors was evaluated in transgenic mice and their litter control partners in a blind trial. All animal maintenance and handling in accordance with Spanish and EU regulations (Directive 2010/63 / EU).

Animals

Experimental procedures involving the use of live mice were carried out in accordance with Spanish and EU regulations (2010/63 / EU), and were approved by the Internal Bioethics Committee of the Institute of Neurosciences and the CSIC. The animals were housed in ventilated cages in a temperature controlled SPF environment, (23 ° C), at a humidity between 40% and 60%, and in a 12-hour cycle of

5

10

fifteen

twenty

25

30

35

light darkness. The mice had acf libitum access to food and water and the cages were changed weekly.

Mouse Line Generation

A tag containing 5 Myc epitopes was added to the grikA rat cDNA, just after the signal peptide (first 20 amino acids). The functionality of the pcDNA3 myc grikA plasmid was tested through electrophysiological recordings when expressed in HEK293 cells. The Myc-grikA construct to generate the transgenic mice was cloned in two consecutive stages. First, Myc-grikA was cloned as an Eco RV-Sma I fragment in plasmid pNN265 that contains an intronic sequence necessary for the proper functioning of the CaMKII promoter (Figure 1). Subsequently, a Not I fragment containing Myc-grikA and the intronic sequence was introduced into plasmid PMM403 which has the CaMKII promoter inserted in pBluescript (Mayford, M., et al. 1996. Transgene Science 274, 1678-1683). The construction was cut with the restriction enzyme Sfi I to eliminate the structure of pBluescript and injected into the pronucleus of fertilized eggs (performed at the Unitat Animáis Transgénics -UAT-CBATEG-, Center for Animal Biotechnology and Gene Therapy, Autonomous University of Barcelona).

Of the nine founders obtained, one was discarded due to integration into the Y chromosome. Myc-grikA expression levels in the F1 generation were analyzed by in situ hybridization, immunohistochemistry and Western blots. Two different lines with similar levels of expression were chosen for further studies.

Western blots

Hippocampal and cortical protein lysates were analyzed in Western blots that were tested with the following primary antibodies: anti-alpha-tubulin mouse (1: 1000, Abcam), anti-Myc mouse (1: 500, Santa Cruz), rabbit anti-GluR6 / 7 and rabbit anti-KA2 (1: 1000, Millipore), rabbit anti-GluR2 / 3 (1: 1000, Novus Biological) and rabbit anti-KA1 (1: 1000: a generous gift from Dr. Melanie Darstein) (Darstein, M., et al. 2003. J. Neurosci. 23, 8013-8019). Antibody binding was detected by a luminescent image analyzer (LAS-1000plus; Fuji) and quantified with Quantity One 1D Analysis Software (Bio-Rad Laboratories). For quantifications, all densities were normalized to the respective tubulin signal.

ojjaiqe odoiBQ

be

eun epeo sauojej 02 9P sajuaja.np sapoipo sop opuezpn ‘pepa ap seueiuas ziS sp sauojej ua uojezpaj as ojuaiwejjodwoo ap SBqanjd sen

ojuaiwejjodwoo ap seqarud

[zi: on ai os

03S], S-1V0010Q111100V01VVV0V011010010VV011V0111V001Q-, S: oÁ | / \ |

[u: on ai

03S], S-V10V1VV0VVV101010VV01V1V0001000101000000V1-, S: q WMO

[01: 0N ai03S]

, S-0101V00V0110010VV000V0101V00V1000VV0110V19110-, S: b WMO S2

iuojaai sepezpn sopuoapnuo6p ap sepuanoas se “| sopej | nsaj

soopiapi uojafnpojd Á opiejed ua uojepuqiij as pyuB ua6 | a ejed sajuaipuadapui

sopuoapnuo6p sop ‘pepp ^ padsa ap iojjuoo ooioq (6S- £‘ V lo / qojnaN Aay fu¡

ZOOZ T 3 sujoiai pue and \ A uapsiM) ajuaweiAajd opuosap as oiuoo sge uoo sbpbojbuj 02

sooypadsa sop! joapnuo6i | o ap sepuos uoo uoo ozpaj as n} is ui uopepuqm en

Wildebeest uopepuqiH

(000U = U) l-V> Hlue oíauoo ap Á (oos: | -) oA | / \ MJue

uojbj ap sodjanoiiue uoo joiq ujajsa / \ / \ ua sopesn so | uojezpue as ‘bjoij \, uopabip gi. eun ap sandsaa sope6! | -N sopueoesoBp so | sopoj jeuiiup ejed oa | dwa as (sqeopia puB | 6u3 M3N frOZOd) d9SB0Nd 9P | Bn6 | pepijueo eun Á esouew ua opiuajuoo oi | B uoo sopuBOBso6no so | JBUiLuna ejed uojBsn as (sqeioia pue | 6ug aaon! 20Z0d)

Hopug ap sapeppn OOS anb o | uoo '{plp-gop ‘822 qojeasay uiejg / ejnoiAeqag '21 -02

| B ia ‘s T‘ sagopo) ajuaiueiAajd ojuosap ei | as anb | en6 | ojsaj as jaseQNd / Hopug 01-

uojoe | jsoo! | 6aG

(blu6! S) esepixojad jod eppnpaj aupzuaqouoiueip Á (sauopjoqen JopaA) 9JN3 03V ap i!> | | opposite ozpns | A as odjanouue □ (SON &% P lB VSa '% 2 lB 001 - X uojui'% U'0 S8d) oanboiq ap uopnps ua (weoqv 000U: U) oA | / \ H} ue o || od ap sodjanoijue uoo g

Oot ^ e aipou e \ ajuejnp uojeqnoui as (lurl os) sauopoas sen% p \ e opjiiapieuijopjed ua aipou e \ epoj ajuauuouajsod opefu

eojuijnbo; sjqounuiu |

5

10

fifteen

twenty

25

30

35

The open field test was carried out in a 50 cm x 50 cm long stage for 30 minutes, recording the behavior with a video tracking system (Panlab, Spain). Differences in distance traveled, rest time, and time in the center and periphery of the stage, were calculated for GluK4 + / + and GluK4over mice in relation to total time.

Labyrinth raised in cross

A metal maze raised 50 centimeters above the ground that contained two dark and closed arms, and both arms open and lit, was used to examine anxiety-like behaviors. The arms were 50x10 centimeters in size with a central area 10x10 centimeters, and the walls of the closed arms were 30 centimeters high. The individual mice were placed in the center of the maze, followed for 10 minutes with a video camera and then returned to their housing cage. The elevated maze was cleaned between trials with a 70% alcohol solution. The time and frequency of visits to the different areas of the maze was evaluated using the Smart Video Tracking System (Panlab, Spain).

Forced swimming test

Transparent plexiglass cylinders, 50 centimeters high and with a diameter of 25 centimeters, were filled to approximately 25 centimeters with tap water at 22-24 ° C, so that the mice were not able to touch the ground or escape . The individual mice are placed in the water for a 6-minute session and recorded on video for later analysis. At the end of each session, the mice were dried with a paper towel and returned to their cage, and the water was replaced for the next test. The immobility time, defined as a lack of active swimming, was noted.

Light / dark test

The test apparatus consisted of two rectangular plexiglass boxes (20x20x20 centimeters) connected by a small hallway (6 centimeters). One was open and illuminated, while the other compartment was dark. Each mouse was placed in the dark compartment and kept in the sand for 10 minutes. The time in which he left the dark compartment for the first time and the time spent in the illuminated compartment was measured by video recording and analysis using Smart software (Panlab, Spain).

5

10

fifteen

twenty

25

30

35

Sucrose Intake Test

Anhedonia was evaluated by controlling sucrose intake. For one week, the animals were provided with two bottles of drinking water, one with tap water and the other with a 2% sucrose solution. After this acclimatization, the mice could choose to drink any of the solutions, sucrose or tap water for a period of 4 days. The intake of water and sucrose solution was measured daily, and the position of the two bottles was changed daily to reduce lateral bias. The preference for sucrose was calculated as a percentage of the volume of sucrose intake over the total volume of fluid intake and average during the 4 days of testing.

RotaRod

We use the RotaRod from Ugo Basile (Italy) for the motor coordination test. One day before the test, the animals were trained on the RotaRod set at 16 rpm until they could remain in it for 30 s. For testing, the RotaRod was set to accelerate from 4 rpm to 40 rpm over the course of 5 minutes, and the latency to fall from the rod was recorded. The test was performed on 4 consecutive days, three times a day.

Social interaction test of the three chambers

This test was carried out as previously described (Yang M., et al. 2011. Curr. Protoc. Neurosci. 56, 8.26.1-8.26.16; Smith SE1, et al. 2011. Sci Transí Mecí 3, 103ra97). Briefly, the social apparatus consists of a rectangular box of three chambers, each measuring chamber 20 centimeters long x 40.5 centimeters wide x 22 centimeters high. The dividing walls were transparent plexiglass, with small openings (10x5 centimeters) that allow access to each chamber. The test consisted of four phases: phase I, a habitation period of 5 minutes, where the mouse moved freely in the central chamber; Phase II consisted of accustoming the mice to the three chambers for 5 minutes with two empty round grid cylinders located in the corner of each of the side rooms; In phase III the mouse sociability was determined for 5 minutes by measuring the time spent by the mouse with freedom of movement in the vicinity and in the housing of the grid box housing containing a first foreign mouse; and, finally, phase IV was the test of preference for social novelty, when a second novel mouse was placed inside the previously empty grid cylinder while the first novel mouse remained inside its grid cylinder. The

5

10

fifteen

twenty

25

30

35

Preference for social novelty was defined as the time in the chamber with the new mouse and the inhalation time of the novel mouse compared to the family mouse. The trajectories were recorded and analyzed with Smart software.

Electrophysiological records

Hippocampal sections were prepared from mice aged 14 to 16 days, as described in detail in other studies (Christiansen et al., 2004, J. Neurosci. 24, 8986-8993). Simply put, the entire brain that contains both hippocampus was placed on the stage of a vibratome slicer and cut to obtain sagittal brain cuts 300 nm thick, and that were continuously oxygenated for at least 1 hour before its use. Electrophysiological records were made from neurons visually identified by IR-DIC microscopy using a 40x water immersion objective. All experiments were carried out at room temperature (22-25 ° C). The sections were continuously perfused with a solution consisting of (in mM) 124 NaCI, 2.69 KCI, 1.25 KH2P04, 4 MgCI2, 2 CaCI2, 26 NaHC03 and 10 glucose (pH 7.3; 300 mOsm), supplemented with drugs (LY303070, APV, picrotoxin) as needed. The EPSCs were caused by electrical impulses applied to the dentate gyrus with a bipolar electrode. Recordings of hermetically sealed whole cells (> 1GQ) were obtained from the cell body of pyramidal CA3 neurons. The electrode patches were made of borosilicate glass and with a resistance of 5-10 MQ when filled with (in mM): 122 CsCI, 8 NaCI, 10 HEPES, 5 EGTA, 10 TEA (pH 7.3, 287 mOsm ), supplemented with 5 mM of QX-314 (Alomone Laboratories, Israel). The membrane potential was set using an Axopatch 200A amplifier (Axon Instruments). Access resistance (8-30 MQ) was monitored regularly during recordings and the cells were rejected if more than 15% had changed during the experiment. The data was filtered at 2 kHz, digitized and stored on a computer with the pClamp software.

In transfected HEK293 cells, electrophysiological experiments were carried out 1 day after transfection with the plasmid. The cells were rapidly perfused using a linear set of glass tubes placed 200-300 microns from the cell body driven by a motorized device under the control of a personal computer.

5

10

fifteen

twenty

25

30

35

The role played by kainate receptors (KARs) in the physiology of the brain is worse understood than that of other glutamate receptors, however, it is now clear that they play an important role in the synapse and maturation of neuronal circuits during the development. To assess the possible role played by the variation in the number of copies (CNVs) of genes encoding the GluK4 subunit in ASDs, the physiological and behavioral consequences of over-expression of gr¡k4 in the low mouse forebrain were evaluated CaMKII promoter control (Figure 1). Two of 9 lines of mice in which the different levels of expression gr¡k4 was evident (GluK4over lines) were selected. In Western blotting, the protein encoded by grk4, GluK4, was overexpressed by 40% in the hippocampus and slightly more than 100% in the neocortex. Other KAR subunits were induced, either in the cortex, but not in the hippocampus (GluK2) or were repressed in the hippocampus (Figure 2a, 2b), probably reflecting a process to compensate for excess GluK4.

The effect of GluK4 overexpression on behavior was assessed by a large battery of behavioral tests, demonstrating significant differences between the GluK4over mice and their GluK4 + / + siblings. Since the diagnosis of autism is based on the deficit in social behavior, social interactions were first looked at. In the three-chamber social interaction test (Nadler, JJ et al. 2004. Genes Brain Behav. 3, 303-314), both GluK4over and wild-type mice interacted more strongly with a mouse than with an object, which indicates a correct level of discrimination of the congeners of inanimate objects (Figure 2c). However, in contrast to normal mice, GluK4over interacted with equally new and familiar mice when faced with a new mouse (Figure 2c, 2d), indicating a deficit in their social interactions, typical of ASDs. Tracking the trajectories of the GluK4over mice revealed a tendency to remain in the chamber occupied by the family mice, although far from them (Figure 2c). In addition, the time spent by the GluK4over in the chamber occupied by any of the mice, excluding the area in close proximity, also revealed that the transgenic animals spent significantly less time in the chamber occupied by the new mouse, tending to remain in the chamber occupied by the family mouse once the scan was complete (Figure 3).

5

10

fifteen

twenty

25

30

35

In children with ASDs, some degree of anhedonia has recently been demonstrated (Chevallier, C., et al 2012. J Autism Dev Disord 42, 1504-1509), that is, the absence of experiencing normally pleasant life events such as eating. Interestingly, GluK4over mice reflect a significant degree of anhedonia compared to normal mice, as evaluated by the lower intake of sucrose (Figure 2f). Given that Anhedonia is a clinical symptom of depression, we subjected GluK4over mice to the forced swimming test. Transgenic animals demonstrated a higher immobility index than normal mice in this test (Figure 2g), which demonstrates depressive behavior. To check the direct involvement of the GluK4 protein, this depressive state was compared with mice deficient for GluK4. KO mice for grík4 spent significantly more time swimming than controls and those that overexpress the transgene (Figure 2g), this shows a clear correlation between the expression of grík4 and the anhedonic state, and suggests a direct role for CNV of grik4 in this behavior disorder. It has also been pointed out that GluK4over mice may experience anxiety, as they avoid open areas. Anxiety was directly tested in three independent tests routinely used to assess such behavior in animals (Lalonde, R. & Strazielle, CJ2008. Neurosa. Methods 171, 48-52): the raised labyrinth in cross, the open field scenario and a dark / light box. In all these tests, GluK4over mice show signs of anxiety. For example, they spent significantly less time in the open arms of the raised labyrinth than their brothers (Figure 4a), were much less willing to explore the center of an open field (Figure 4b) and spent more time in the dark area than in the light in the light / dark test (Figure 4c). In general, GluK4over mice spent less time moving than their siblings, traveling shorter distances in all tests. However, this was not due to any motor impairment, since transgenic and wild-type animals perform equally well in rota-rod tests (Figure 5). In contrast, the increase in sedentary periods of mice that overexpress GluK4 could reflect a lack of motivation.

In light of the above, it was questioned whether overexpression of the GluK4 receptor subunit could influence the transfer of normal information in the brain. Neural KARs have been studied extensively in the CA3 field of the hippocampus, where they have been located in pre-and post-synaptic positions. In addition to memory, the

5

10

fifteen

twenty

25

30

35

Hippocampus has been associated with aspects of emotional processing, in particular, with anxiety. Therefore, the flow of information through the circuit was evaluated in the trisynaptic circuit of the hippocampus, in obtaining recordings in vivo using a vertical set of electrodes in anesthetized animals. The electrical stimulation supplied to the perforating pathway, the entry into the trisynaptic circuit, evoked similar electrophysiological responses through genotypes at the level of the first synapse of the dentate gyrus (DG). Consequently, no differences were found in the input / output curves measured at the level of the dendrites or the soma (Figure 6). However, the field excitatory postsynaptic potential (fEPSP) evoked in CA1 through trisynaptic activation appears to be significantly higher in GluK4over mice than in normal mice. Indeed, the plot of the population peak amplitude of the DG (PS) versus the slope of fEPSP in CA1 (i.e., the entrance and exit to the trisynaptic circuit, respectively) revealed, in addition, a change in the gain of this circuit. Subsequently, the synoptic transmission of the mossy fibers to the pyramidal neurons CA3, the second synoptic relay in the trisynaptic circuit, where GluK4 is strongly expressed and appears to play critical physiological roles, was evaluated. Post-synaptic excitatory KAR-mediated currents (EPSCKAR) in mice that overexpressed GluK4 have been 39% larger than in normal mice and with 13% faster deactivation times (Figure 7a and 7b). This result confirms that the excess GluK4 protein expressed participates in functional KARs. The analysis of mEPSCs mediated by AMPA receptors at this synapse revealed a higher frequency and amplitude of mEPSCs in GluK4over mice (Figure 7d), and the evoked EPSCs were also consistently larger (Figure 7e). On the contrary, the facilitation by pairs of pulses, a kind of short-term plasticity that is a characteristic of these synapses, was significantly affected (Figure 7f). Together, these data imply that the transfer of information at these critical synapses is altered in mice that overexpress the GluK4 subunit of the KARs.

The phenotype of the GluK40Ver mice suggests that this protein may be involved in the pathogenesis of autism spectrum disorders, providing a model for the mechanistic investigation of the disease. It is important to highlight that, in the present study, it is demonstrated that a moderate increase in protein levels (30-50%) in animals that overexpress it, however, is associated with a deep functional penetrance, altering the transfer of information and

5

10

fifteen

twenty

25

30

35

integration into a robust hippocampus network. Autism is considered a disease of brain development and there is strong evidence that KARs influence the development of neural networks. For example, the activity of the immature network seems to influence the development of synoptic connectivity and the KARS play a critical role in the activity of the stimulation network in both the hippocampus in development and adults. In addition, the tonic activity of the KAR influences the maturation and elongation of neurites. Therefore, it is conceivable that the altered synoptic transmission through generalized neural networks arises as a consequence of the excess of GluK4 in particular glutamatergic synapses during the formation and maturation of the network, which leads to a permanent change in the synoptic gain.

Currently, ASDs are believed to represent a continuum of the same disease with varying degrees of severity, and that there is genetic overlap between ASDs and other neurodevelopmental and neuropsychiatric endophenotypes. For example, ASDs are associated with clinical indications of anxiety. Therefore, it is quite remarkable that single-gene CNVs can reproduce some endophenotypes seen in autism, and somehow mimic the situation in humans with an ASD. Such alterations to gene expression in mice reflect one of the molecular mechanisms that underlie abnormal behaviors in humans. Therefore, it is possible to use this mouse to directly test specific behavioral treatments related to autism.

Given the clear phenotypes of depression, anxiety and anhedonia that these mice present, it has been wanted to verify if a well known antidepressant and / or anxiolytics would be effective in these phenotypes as a proof of concept in the use of this mouse model in the search for New therapeutic drugs It was decided to try fluoxetine and thiapentine. Fluoxetine is also known by the trade names Prozac, Sarafem, Ladose and Fontex, among others, and is an antidepressant of the serotonin reuptake inhibitor (SSRI) class and is used for the treatment of major depressive disorder (including pediatric depression), obsessive -compulsive disorder (in adults and children), bulimia nervosa and panic disorder. Thianeptin, which brand names are Stablon, Coaxil, Tatinol and Tianeurax, is a medication that is mainly used for the treatment of major depressive disorders; Chemically it is a tricyclic antidepressant (TCA), but it has different pharmacological properties. Although it can serve as a

5

10

fifteen

twenty

25

serotonin reuptake promoter, the most recent research indicates that thianeptin produces its antidepressant effects through indirect alteration of the activity of AMPA and NMDA glutamate receptors (McEwen, BS et al., 2010, Molecular Psychiatry 15: 237 -49; Zhang H. Et al., 2013, Molecular Psychiatry 8, 471484).

The wild-type and GluK4over mice were injected with doses equivalent to those of their use in humans and the effect on the symptoms of anhedonia, anxiety and depression evaluated after 30 days of treatment. Fluoxetine had no action on depression in the GluK4 animal model of depression according to the evaluation of the forced swimming test (Figure 8). However, thianeptin had a strong antidepressant effect in these mice, restoring immobility control values in this assay (Figure 8). Similarly, fluoxetine worsened anhedonia in WT mice, a behavior easily detected in autism. On the other hand, thianeptin restored the control values of sucrose intake in GluK4over mice, indicating a strong inhibition of anhedonia in this autism model (Figure 9). The effect on anxiety was assessed by the elevated cross labyrinth test and the open field test. Thianeptin had no effect in the time spent by GluK4over animals in the center of the open field or in the number of entries in the open arm of the raised labyrinth, indicating a lack of anxiolytic effect. However, thianeptin normalized the depressive locomotor activity in GluK4over mice (Figure 10), indicating some therapeutic value in this mouse model. In the open field test, fluoxetine not only aggravated anxiety in anxious GluK4over mice, but also produced some anxiety in normal mice (Figure 11). All these data indicate the disparity of action of known antidepressants and anxiolytic drugs depending on the etiology of these diseases, indicating that the search for new and more appropriate drugs should continue to treat these disorders in a more refined way.

Claims (27)

5 10 fifteen twenty 25 30 35 1. A non-human transgenic animal that comprises stably integrated into its genome a heterologous nucleotide sequence encoding the GluK4 protein, in which said nucleotide sequence is expressed in the animal's anterior brain. 2. A non-human transgenic animal according to claim 1, wherein the GluK4 protein comprises an amino acid sequence with a sequence identity of at least 95% at SEQ ID NO: 1. 3. A non-human transgenic animal according to claim 2, wherein the GluK4 protein comprises the amino acid sequence SEQ ID NO: 1. 4. A non-human transgenic animal according to any one of claims 1 to 3, wherein the nucleotide sequence encoding the GluK4 protein comprises the nucleotide sequence SEQ ID NO: 2. 5. A non-human transgenic animal according to any one of claims 1 to 4, wherein the GluK4 protein is the rat GluK4 protein. 6. A non-human transgenic animal according to claim 5, wherein the nucleotide sequence is expressed in the hippocampus and / or in the neocortex. 7. A non-human transgenic animal according to any one of claims 1 to 6, wherein the heterologous nucleotide sequence encoding the GluK4 protein is operably linked to an expression regulatory sequence. 8. A non-human transgenic animal according to claim 8, wherein the expression regulatory sequence is a promoter and / or an enhancer. 9. A non-human transgenic animal according to claim 9, wherein the promoter is a specific promoter of the Central Nervous System. 10. A non-human transgenic animal according to claim 10, wherein the promoter is the CaMKII alpha promoter or the promoter of the grk4 gene. 5 10 fifteen twenty 25 30 35 11. A non-human transgenic animal according to any one of claims 1 to 10, comprising at least 2 copies of the heterologous nucleotide sequence encoding the GluK4 protein. 12. A non-human transgenic animal according to any of claims 1 to 11, wherein the animal is a mammal, preferably selected from the group consisting of monkey, pig, rat and mouse. 13. A method for the detection, discovery, identification, evaluation and / or validation of a compound for the treatment of an autism spectrum disorder, anxiety and / or depression, comprising (a) administering a candidate compound to an animal Non-human transgenic according to any one of claims 1 to 12 and (b) determining the effect of the test compound on the behavior of said non-human transgenic animal or, alternatively, determining the level of expression of the GluK4 protein. 14. Use of a transgenic animal according to any of claims 1 to 12, in the screening, discovery, identification, evaluation and / or validation of a compound or therapy to treat autism spectrum disorders, anxiety and / or depression. 15. Use of the non-human transgenic animal according to any of claims 1 to 12 as an animal model for autism spectrum disorders, anxiety and / or depression. 16. A method of producing a non-human autism animal model comprising the expression in the anterior brain of the animal of a heterologous nucleotide sequence encoding the GluK4 protein, in which the heterologous nucleotide sequence is stably integrated in the genome of the non-human animal. 17. Method according to claim 16, wherein the protein comprises GluK4 amino acid sequence with a sequence identity of at least 90% at SEQ ID NO: 1. 5 10 fifteen twenty 25 30 35 18. Method according to claim 17, wherein the GluK4 protein comprises the amino acid sequence SEQ ID NO: 1. 19. A method according to any of claims 16 to 18, wherein the nucleotide sequence encoding the GluK4 protein comprises the nucleotide sequence SEQ ID NO: 2. 20. Method according to any of claims 16 to 19, wherein the GluK4 protein is the rat GluK4 protein. 21. A method according to claim 20, wherein the nucleotide sequence is overexpressed in the hippocampus and / or in the neocortex. 22. A method according to any of claims 16 to 21, wherein the heterologous nucleotide sequence encoding the GluK4 protein is operably linked to an expression regulatory sequence. 23. The method of claim 22, wherein the expression regulatory sequence is a promoter and / or an enhancer. 24. Method according to claim 23, wherein the promoter is a specific promoter of the Central Nervous System. 25. The method of claim 24, wherein the promoter is the CaMKII alpha promoter or the grik4 gene promoter. 26. A method according to any of claims 16 to 25, comprising introducing at least 2 copies of the heterologous nucleotide sequence encoding the GluK4 protein into the genome of the non-human animal. 27. A method according to any of claims 16 to 26, wherein the animal is a mammal, preferably selected from the group consisting of monkey, pig, rat and mouse.