WO2010126389A1

WO2010126389A1 - Process for treating neural stem cells based on ampakines and/or other modulators of ionotropic glutamate receptors, compositions thereof and their use in cns conditions

Info

Publication number: WO2010126389A1
Application number: PCT/PT2010/000020
Authority: WO
Inventors: João José OLIVEIRA MALVA; Clarissa De Sampaio Schitine; Fabienne Agasse; Sara Alves Xapelli; Ana Paula Pereira Da Silva Martins; Ricardo Augusto De Mello Reis
Original assignee: Universidade De Coimbra
Priority date: 2009-04-28
Filing date: 2010-04-28
Publication date: 2010-11-04
Also published as: PT105083A

Abstract

The present invention refers to a process for treating neural stem cells based on AMPAkines and other modulators of ionotropic glutamate receptors. These compounds induce a strong proliferative and proneurogenic action and an effect in neuronal differentiation when applied to SVZ cell cultures. Therefore, in another aspect of the present invention, compositions based on AMPAkines and other modulators of ionotropic glutamate receptors are described to be used as inducers of neuron production and/or differentiation when applied to SVZ cell cultures. Further, the present invention also refers to compositions comprising neural stem cell cultures treated with AMPAkines and/or other modulators of ionotropic glutamate receptors may be used as a drug on the treatment, prevention and/or to enhance CNS conditions as such brain disease and/or non-pathological brain conditions. Therefore, the present invention is applicable in the pharmacology and medical areas.

Description

DESCRIPTION

PROCESS FOR TREATING NEURAL STEM CELLS BASED ON AMPAKINES

AND/OR OTHER MODULATORS OF IONOTROPIC GLOTAMATE RECEPTORS,

COMPOSITIONS THEREOF AND THEIR USE IN CNS CONDITIONS

Technical field of the invention

The present invention refers to a process for the treatment of neuronal stem cells with AMPAkines and/or other modulators of ionotropic glutamate receptors, compositions comprising such neuronal stem cells and their use to treat, prevent and/or enhance CNS conditions. Therefore, the present invention may be applied to pharmaceutical and medical areas.

Background of the invention

The mammalian hippocampus encloses an important neurogenic niche, located in the subgranular zone of the dentate gyrus. In this brain region, neural stem cells self-renew, divide, differentiate and migrate locally into the granular cell layer, where new cells are integrated (Gray and Laskowski, 2007; Kempermann et al, 2008) .

This important neurogenic resource plays an important physiological role in the hippocampus, and consequently dysfunctional neurogenesis results in major behavioural dysfunctions related with memory circuits. At least two important evidences support the role of hippocampal neurogenesis in memory and learning: 1- the rate of neurogenesis determines learning performance and memory; 2- learning tasks enhance neurogenesis and new neuron survival (Abrous and Wojtowicz, 2008) .

Interestingly, it has been demonstrated that depressive behaviour is associated with shrinkage of the limbic system, with a particular impact in the hippocampus. As for learning and memory, there is also a close association between depression and neurogenesis. Accordingly, depression induces neuronal degeneration and decreased hippocampal neurogenesis, whereas antidepressants tend to increase neurogenesis rate (Sahay et al, 2008) .

Additionally, following injury, the production of new cells, increases in the neurogenic niches, especially in the subventricular zone (SVZ) , allowing cells to migrate and replace dead counterparts. Therefore, the capacity of stem/progenitor cells to proliferate and generate repairing cells is yielding great expectations regarding their use in brain repair.

Recent evidences show that neurogenesis is strongly influenced by neuronal activity, involving synaptic and non- synaptic mechanisms (Kempermann et al, 2004; Ge et al, 2007; Jang et al, 2008) .

Particularly striking is the reported stimulatory effect of GABA and glutamate in hippocampal and SVZ neurogenesis (Tozuka et al, 2005; Platel et al, 2008) . The orchestrated spatio-temporal action of the excitatory effects, due to GABAA receptor activation and ionotropic glutamate receptors (Kempermann et al, 2008; Jang et al, 2008), set the driving force that moves cell differentiation from neural progenitors into post-mitotic granular cells and SVZ derived neuroblasts.

AMPA receptors are members of ionotropic non-NMDA receptor family, with a central role in post-synaptic depolarization and fast excitatory transmission. Functional AMPA receptors are assembled in tetramers of subunits (GluRl-4), forming dimmer-dimmer complexes with a functional receptor operated channel. Important post-transcriptional modifications including RNA editing and alternative splicing of the variants flip/flop, as well as quality control of subunit assembly taking place at the endoplasmic reticulum, contribute to existent diversity of functional receptors. These modifications are especially relevant concerning membrane surface expression, gating motion and desensitization properties (Greger et al, 2007) .

AMPAkines (Formula I) are a structurally diverse family of small molecules that freely cross the blood-brain barrier and work as positive allosteric modulators of AMPA receptors.

(Formula I)

AMPAkines have been reported as potential pro-cognitive enhancers based on in vitro and in vivo studies (Wezenberg et al., 2007) and their role in synaptic responses was first disclosed on the document W09402475.

Ampakines work by allosterically binding to particular receptors in the brain, called AMPA-type glutamate receptors, i.e. by up modulating neural activation and transmission in neurons containing glutamatergic receptors. This boosts the activity of glutamate, a neurotransmitter, and makes it easier to encode memory and to learn.

AMPAkines have no agonist or antagonist effects; instead, they stabilize the receptor in its channel-open state following the binding of endogenous glutamate (Lynch and Gall, 2006) .

The reported cellular/molecular/network effects attributed to AMPAkines in mice include: 1- enhancing synaptic transmission; 2- lowering the threshold and increase in long-term potentiation magnitude; 3- increased expression of BDNF; 4- increased brain activity in structures associated with behavioural demand (Lynch and Gall, 2006; Lynch, 2006) .

Hyperactivation of glutamate receptors is a major cause of seizure activity and excitotoxic brain damage. Interestingly, in spite of the positive modulatory action of AMPA receptors by AMPAkines, an accumulating body of evidence has shown that these molecules are not toxic (Lynch, 2006) . Members of this drug family (BPD 29, CX554, CX516, CX546, CX 614, CX 717, Farampator) have shown a broad range of potential therapeutic applications, including: memory retention and memory acquisition; depression; sleep deprivation; attention-deficit hyperactivity disorder; schizophrenia, and others (Lynch, 2006) . In summary, AMPAkines contribute to stimulate the brain's network activity.

On the other hand, experimental data and clinical trials indicate that AMPAkines may work as potent cognitive enhancers, including in humans, by reinforcing hippocampal network activity.

However, at network level, the processes involved in AMPAkines-induced alertness, antidepressant and putative cognitive enhancing properties have not been clearly identified. The fashionable thinking involves the stimulation and increased efficiency of synaptic neurotransmission in hippocampal circuits.

It has been proposed as a treatment for Rett syndrome, after favorable testing in an animal model (Ogier M, Wang H, Hong E, Wang Q, Greenberg ME, Katz DM (October 2007) .

Drugs based on AMPA modulating activity, such as the ones of racetam family, have been described in prior art for treating neurological pathologies. However, their mode of action and effects stills need to be fully clarified.

WO9921422 describes the use of some of these drugs for the treatment of schizophrenia and related disorders. However, such compositions are not able to induce proliferative and/or proneurogenic action such composition has been associated with improuved synaptic efficiency and long-term potentiation on the SVZ and thus they may only be able to act to a restricted neurologic pathologies.

WO2005072345 discloses drugs to enhance the therapeutic effects of an AMPA receptor potentiator by co-administration of an acetylcholinesterase inhibitor with said potentiator. However, such compositions also show the same disadvantages as referred above.

"Proneurogenic action" in the sense of the present invention means the capacity of a neural stem cell resource to generate differentiated and functionally mature neural cells, including new neurons.

It is now disclosed that the stimulation of neural stem cells, derived from the SVZ by AMPAkines also involves increase in proliferation and neurogenesis. Therefore, pharmaceutical compositions based on neural stem cells treated with AMPAkines are able to work as pro-cognitive and antidepressant modulators by increasing neurogenesis, and therefore they play a central role in new cellular and pharmacological-based strategies to treat and/or prevent central nervous system (CNS) diseases and also to be used in increasing and/or maintaining brain cognitive abilities. Such compositions are able not only to maintain the effects of the compositions of the prior art based on AMPAkines but also to improve responses of the CNS in pathological and non-pathological conditions.

General description of the invention

The present invention refers to a process for the treatment of neural stem cells with AMPAkines and/or other modulators of ionotropic glutamate receptors. Compositions comprising said treated stem cells are also disclosed and also their use to prevent, enhance or treat CNS conditions.

Glutamate has been reported as a signal important for the control of neuroblasts proliferation on stem cell niches in the brain (Platel et al., 2008).

In order to test the hypothesis that AMPA activation/modulation of SVZ neurospheres cultures lead to an increase in the number of proliferating cells, SVZ cultures were incubated with BrdU to investigate the effects of AMPA and AMPAkine CX546 on cell proliferation. A Brdϋ staining was performed on SVZ cell cultures exposed to AMPA.

As shown in figure 1, SVZ neurospheres grown under proliferative or differentiating conditions express AMPA receptors as revealed by the expression of GIuRl and GluR2 and AMPA 5μM induced an approximately fourfold increase in the percentage of BrdU positive cells on SVZ cultures treated for 48h (Fig.2) .

Addition of CX546 had a similar impact on SVZ cultures in terms of increase in the number of BrdU positive cells, since 5 μM CX546 induced a two-fold increase in BrDU+ cells, while at 50μM CX546 shows a 4 fold increase and CX546 150μM increases 6 times the number of BrDU+ cells compared to untreated cultures. These data indicate that AMPA or AMPAkine CX546 enhances BrdU incorporation in neural progenitor cells of the SVZ in a dose dependent manner.

Concerning cell differentiation, addition of AMPA 5μM resulted in approximately two-fold increase in the percentage of NeuN lmmunoreactive cells (a neuronal specific nuclear protein present on mature neurons) , compared with control cultures. SVZ cell cultures exposed to CX546 (50 and 150μM) showed a two-fold and a three-fold increase in the percentage of NeuN positive cells (figure 3) .

In order to functionally evaluate neuronal differentiation in SVZ cultures, the method previously described by Agasse et al, 2008a; WO2008100168 and based on single cell [Ca2+]i variations in response to the presence of KCl or histamine was used.

Membrane depolarization of neuronal cells following exposure to high concentrations of KCl leads to the opening of voltage sensitive calcium channels and massive influx of calcium into the cytoplasm (Ambrόsio, et al, 2000) . However, stimulation with histamine triggers an increase in [Ca2+]i in immature SVZ cells (Agasse et al, 2008a) .

This reliable method allows the fast identification of a ratio of response to histamine or KCl exposure (Hist/KCl) . This ratio differs significantly between cell type groups, with a low Hist-KCl ratio (below 0.8) being characteristic of SVZ derived neurons (Agasse et al, 2008a) . Thus, to investigate if AMPA and CX546 could promote SVZ cells differentiation into functional neurons, 6-8 day SVZ neurospheres were differentiated on poly D-Lysine coverslips for 7 days in the presence of AMPA (100 nM to 50μM) or AMPAkine CX546 (500 nM to 150μM) .

As shown in Figure 4 and figure 6, SVZ cell cultures treated with AMPA for 7 days increased up to threefold, in a dose- dependent manner, the percentage of neuronal like responding SVZ cells. Accordingly, CX546 (500 nM to 150μM) treatment increased up to twofold the percentage of neuronal-like cells in SVZ cultures (figure 5 and figure 6) .

In contrast, control, non-treated cultures showed a predominant immature-like profile, characterized by an increase in [Ca2+]i in response to histamine but a small response or no response to KCl stimulation. The differentiation profile of SVZ cell cultures induced by the AMPA or the AMPAkine3 CX546 was similar to the previously reported proneurogenic effect of neuropeptide Y (Agasse et al., 2008b) or low concentrations of tumor necrosis factor alpha (Bernardino et al., 2008).

The next assay was performed in order to test if AMPA and AMPAkine CX546 could activate Stress-Activated Protein Kinase (SAPK) -jun amino terminal kinase (JNK) pathways in SVZ cultures since it is known that these signaling pathways are involved in neurogenesis, (Amura et al, 2005; Agasse et al., 2008b; Bernardino et al., 2008) and are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. As shown in figure 7, addition of AMPA lμM or AMPAkine CX546 5μM for 6 hours increased the number of ramifications per neurosphere and the total length of JNK immunoreactive tau- positive ramifications (figure 7), when compared to control SVZ cultures.

Altogether, these results show that activation and/or modulation of AMPA subtype of glutamate receptor increase axonogenesis, differentiation and functional maturation of neurons in SVZ cultures. As mentioned before, AMPA receptors activation may trigger neuroblasts proliferation and differentiation by increasing the intracellular calcium concentration (a key intracellular messenger with a central role in cell signalling for important functions, like cell division) and membrane depolarization / cell excitability.

The ionotropic properties of AMPA receptors and the selective permeability of AMPA receptors lacking GluR2 subunits to calcium ion contribute to hypothesize that AMPA receptor activation my play a central role in cell proliferation and functional maturation of neurons.

Therefore, compositions based on AMPAkines are interesting to modulate the gate opening properties of AMPA receptor channels. By increasing the open probability of AMPA receptor channels, AMPAkines can selectively increase the excitability of AMPA receptors exposed to the endogenous local and transient release of endogenous glutamate, increasing neural progenitors excitability, but without significant toxic properties.

1. Process for -the treatment of stem cells

SVZ cells are obtained from 1- to 3-day-old C57B1/6 donor mice and brains are removed following decapitation and placed in HBSS solution (Gibco, Rockville, MD) .

Fragments of SVZ are dissected out of 450 μm thick coronal brain sections, digested in 0.025% trypsin and 0.265 mM EDTA (Gibco) , and dissociated by gentle trituration with a PlOOO pipette. The resulting cell suspension is then diluted in serum-free culture medium (SFM) composed of Dulbecco's modified eagle medium (D-MEM/F12 Gluta-MAX^-I, Gibco) supplemented with 100 U/mL penicillin, 100 μg/ml streptomycin (Gibco) , 1% B27 (Gibco), 10 ng/mL epidermal growth factor (EGF; Gibco), and 10 ng/mL basic fibroblast growth factor (FGF-2, Gibco) .

Single cells are then plated on uncoated Petri dishes at a density of 3000 cells/cm2. The resulting neurospheres are to develop in a 95% air-5% C02 humidified atmosphere at 37⁰C.

Six to eight days after plating, the neurospheres are then collected with a Pasteur pipette and seeded onto poly-D- lysine coated glass cover slips placed into 12-well cell culture plates for calcium imaging experiments or 24- well cell culture plates for immunocytochemistry, and then covered with 1 mL or 500 μL, respectively, of SFM devoid of growth factors.

2. Evaluation of neuronal differentiation

In order to evaluate the neuronal differentiation in SVZ cell cultures, neurospheres are developed over 7 days at 37 °C in the absence or presence of AMPA 1, 5 and 50μM or AMPAkine Cx546 5, 50 and 150μM (both from Tocris) .

3. Single cell calcium imaging

To determine the differentiation pattern of SVZ cells, variations of intracellular calcium levels following stimulation with 50 mM KCl and 100 μM histamine (Sigma, St. Louis, MO) are analysed, as reported elsewhere (Agasse et al., 2008a) . KCl-depolarization causes the increase of the intracellular calcium levels in neurons whereas stimulation with histamine leads to the increase of intracellular calcium levels in stem/progenitor cells. SVZ cell cultures are loaded for 40 min at 37⁰C with 5 μM Fura-2 AM (Molecular Probes, Eugene, OR), 0.1% fatty acid free BSA, and 0.02% pluronic acid F-127 in Krebs (132 mM NaCl, 1 mM KCl, 1 mM MgC12, 2.5 mM CaC12, 10 mM glucose, and 10 mM HEPES [pH 7.4]) .

After a 10-min post-loading period at room temperature the glass cover slip is mounted on RC- 20 chamber in a PH3 platform (Warner Instruments, Hamden, CT) on the stage of an inverted fluorescence microscope Axiovert 200 (Carl Zeiss, Gδttingen, Germany) .

Cells are continuously perfused with Krebs solution and stimulated by applying 100 μM histamine or high potassium Krebs solution (containing 50 mM KCl, isosmotic substitution with NaCl) . Solutions are added to the cells by a fast- pressurized (95% air, 5% CO2 atmosphere) system (AutoMate Scientific, Berkeley, CA) .

2+ The intracellular calcium concentration ([Ca ]i) is evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 ms) at 340 nm and 380 nm, using a Lambda DG4 apparatus (Sutter Instruments, Novato, CA) and a 510 nm bandpass filter (Carl Zeiss) before fluorescence acquisition with a 4Ox objective and a Coll SNAP digital camera (Roper Scientific, Tucson, AZ) . Acquired values are processed using the MetaFluor software (Universal Imaging, Downingtown, PA) . Histamine/KCl values for Fura-2 ratio are calculated to determine the extent of neuronal maturation in cultures. 4. Iππaunocytochemical evaluation

After fixation, for 1 h in 4% paraformaldehyde, cells are permeabilized and non-specific binding sites were blocked for 1.5 h with 0.25% Triton X-IOO (Sigma) and 6% bovine serum albumin (BSA, Sigma) dissolved in PBS. Cells are then subsequently incubated overnight at 4⁰C with the mouse monoclonal anti-NeuN antibody (1:200, Chemicon) rabbit polyclonal antibody anti-phosphorylated form of stress- activated protein kinase (anti-P-SAPK-cjun-NH2-terminal kinase (JNK) (1:100) and mouse monoclonal anti-tau (1:500) (Cell Signaling Technology, Danvers, MAT) .

Thereafter, the cover slips are rinsed in PBS and incubated for 1 h at room temperature with the appropriate secondary antibodies: goat anti-mouse Alexa Fluor 594 antibody (1:200), anti rabbit Alexa Fluor 488, (1:200) (all from Molecular Probes) . After rinsing with PBS, cell preparations are incubated 5 min at room temperature with Hoechst 33342 (2 μg/mL, Molecular Probes) in PBS containing 0.25% BSA for nuclear staining.

Finally, the preparations are mounted using Dakocytomation fluorescent medium (Dakocytomation, Carpinteria, CA) . Fluorescence images are then re recorded using a digital camera coupled to an Axioskop microscope (Carl Zeiss) .

5. Proliferation Assay

To investigate the effect of AMPA and AMPAkine Cx 546 on cell proliferation, SVZ cells are exposed to lOμM 5-bromo- 2 " -deoxyuridine (BrdU) (Sigma-Aldrich) for the last 4 hours of each AMPA or AMPAkine treatment. Then, cells are fixed in 4% PFA for 30 minutes and rinsed for 30 minutes in 0, 15M PBS, at room temperature. Brdϋ is then unmasked and labelled following successive passages in l%Triton x-100, ice-cold 0,1M HCl at 37°C and in borate buffer (0,1M, Na2B4O7.10H20, pH8,5) and incubation of the mouset anti BrdU antibody attached with IgG labelled Alexa Fluor 594 (1:200, Sigma) overnight at 4⁰C .

Nuclei counterstaining and mounting are performed as described previously.

€. Data analysis and statistics

In all experiments, the experimental condition is run at least in triplicate. For SCCI experiments, the cell percentage was calculated on the basis of one field per each coverslip containing about 100 cells. Percentage of NeuN immunoreactive cells are calculated from cell counts in five independent fields in each coverslip with a 4Ox objective (about 200 cells per field) . Because no significant differences were found between controls, the corresponding data were pooled and expressed as mean ± SEM. Statistical significance of differences was examined by one-way ANOVA followed by the Bonferroni posttest for multiple comparisons or unpaired t-test.

In one embodiment of the present invention, a process for the treatment of SVZ cell cultures with AMPAkines and/or and other modulators of ionotropic glutamate receptors is described. In another embodiment of the present invention, compositions comprising SVZ cell cultures treated with AMPAkines and/or and other modulators of ionotropic glutamate receptors, according with the process of the present invention are described.

Other modulators of ionotropic glutamate receptors may be selected from the group of: (±) -2-Amino-4-phosphonobutyric acid solid, (±)-AMPA hydrobromide, (±)-Ketamine hydrochloride, (+)-MK-801 hydrogen maleate, (2S₁ AR) -4- Methylglutamic acid, 1-Aminocyclopropanecarboxylic acid hydrochloride, 1-Naphthylacetyl spermine trihydrochloride, 2, 6-Difluoro-4- [2- (phenylsulfonylamino) ethylthiojphenoxy- acetamide, 6, 7-Dichloroquinoxaline-2, 3-dione, D- -Homocysteinesulfinic acid, DL-2-Amino-5-phosphonopentanoic acid, DL-2-Amino-7-phosphonoheptanoic acid, L-Cysteine hydrochloride monohydrate, L-Homocysteic acid, ATPO, ATPA, CNQX-HBC complex, CX546,, Cyclothiazide, W- (3,3- Diphenylpropyl) glycinamide, PEAQX tetrasodium hydrate, γ-D- Glutamylaminomethylsulfonic acid and others.

Compounds of the racetam family may also be comprised in the sense of the present invention, such as Piracetam, Oxiracetam, Aniracetam, Pramiracetam, Phenylpiracetam, Etiracetam, Levetiracetam, Nefiracetam, Nicoracetam, Rolziracetam, Nebracetam, Fasoracetam, Imuracetam, Coluracetam, Dimiracetam, Brivaracetam, Seletracetam, Brivaracetam, Coluracetam, Rolipram and others.

The stem cell used in the sense of the present invention may be embrionary and/or adult stem cells.

Other active substances may be added to the basic composition in order to enhance and/or potentiate the effect of the AMPAkines and/or other modulators of ionotropic glutamate receptors.

Therefore, in another embodiment of the present invention, the compositions based on SVZ cells treated with AMPAkines and/or other modulators of ionotropic glutamate receptors may include other active substances, such as active substances with neurological effect and/or antibiotics. These active substances may be selected from the group comprising: haloperidol, fluphenazine, perphenazine, chlorpromazine, molidone, pimozide, trifluperazine, thioridazine, clozapine, risperidone, olanzepine, sertindole, ziprasidone, seroquel, zotepine, amisulpride, iloperidone and others. In this sense compounds with acetylcholinesterase inhibitor effect may also be selected.

Furthermore, the said compositions may also include pharmaceutically acceptable vehicles, carriers, excipients and/or other substances commonly accepted and used in pharmacology, such as colorants, preservatives, stabilizers, flavourings, etc. adequately selected for the chosen method of administration.

In another embodiment of the present invention, said compositions may be presented in the form of tablets, capsules, ampoules, liquid and gel compositions and/or controlled release formulations.

The SVZ cell compositions of the present invention may be used to prevent, repair and/or enhance CNS conditions. Such conditions comprise not only the known pathological conditions but non-pathological conditions such as the enhancement of cognitive abilities, such as for maintaining and/or increase memory.

Said pathological conditions, in the sense of the present invention, include chronically conditions such as chronic depression for example.

Other pathologic situations, included in the sense of the present invention, are cognitive conditions, acute depression, psychiatric dysfunctions, schizophrenia, neurodegenerative conditions such as Parkinson and Alzheimer diseases, brain vascular accident and/or in other neuropathic conditions where neuronal loss situations occur or may occur.

Non-pathological conditions comprise, for example, situations where the enhancement of cognitive performance is sought, such as maintaining brain vitality, memory, etc.

The compositions comprising steam cells of the present invention may also be used in transplant techniques to repair CNS conditions and/or injuries, to prevent loss of cognitive capacities and/or to enhance such abilities related to neuronal performances.

In another embodiment of the present invention is described the use of AMPAkines-based compositions for the treatment of neural stem cells.

Description of the figures Figure 1: SVZ neural stem cell cultures express AMPA receptor subunits GIuRl and GluR2 in proliferation (top row) and differentiation conditions (bottom row) . Top row confocal microscopy in cytospin-treated neurospheres. Bottom row - neurospheres were allowed to differentiate in the presence of poli-D-lisine and in the absence of growth factors .

Figure 2: AMPA and the AMPAkine CX546 stimulate cell proliferation in SVZ neural stem cell cultures in differentiation conditions. Blue - staining with Hoechst 33342; Red - Anti BrdU immunostaining of diving cells.

Figure 3: AMPA and the AMPAkine CX546 increase neuronal differentiation in SVZ neural stem cell cultures grown in proliferative conditions. Neuronal differentiation was evaluated by quantifying the number of cells immunopositive for the neuronal marker NeuN.

Figure 4 : The exposure of SVZ neural stem cell cultures to AMPA results in a robust proneurogenic effect. This AMPA receptor-mediated proneurogenic effect is sensitive to the inhibition with GYK52466 compound. The exposure of AMPA induces the increase in the number of KCl-responding cells, with a parallel decrease in the percentage of cells responding to histamine. The inhibition of AMPA receptors with GYK52466 compound changed the profile of the cell population response to a similar pattern compared with control. Figure 5: The exposure of SVZ neural stem cell cultures to AMPAkines results in a robust proneurogenic effect. This AMPA receptor-mediated proneurogenic effect is sensitive to the inhibition with GYK52466 compound.. The exposure to AMPAline induces the increase in the number of KCl- responding cells, with a parallel decrease in the percentage of cells responding to histamine. The inhibition of AMPA receptors with GYK52466 compound changed the profile of the cell population response to a similar pattern compared with control .

Figure 6: The exposure of SVZ neural stem cell cultures to AMPA or to the AMPA receptor modulator AMPAkines, results in a robust neuronal differentiation. The exposure of AMPA induces the increase in the number of KCl-responding cells, with a parallel decrease in the percentage of cells responding to histamine.

Figure 7: The incubation of SVZ neural stem cell cultures with AMPA or AMPAkine CX546, for 6h, results in the increase of the number of JNK+-ramifications migrating out of the neurosphere. Moreover, a robust increase in the total length of labelled JNK structures was also observed.

EXAMPLES

Example 1 - Establishment of SVZ cell cultures SVZ cells were obtained from 1- to 3-day-old C57B1/6 donor mice. Brains were removed following decapitation and placed in HBSS solution.

Fragments of SVZ were dissected out of 450 μm thick coronal brain sections, digested in 0.025% trypsin and 0.265 mM EDTA, and dissociated by gentle trituration with a PlOOO pipette .

The cell suspension was diluted in serum-free culture medium (SFM) composed of Dulbecco' s modified eagle medium (D-MEM/F12 Gluta-MAX™-I, Gibco) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco) , 1% B27 (Gibco) , 10 ng/mL epidermal growth factor (EGF; Gibco), and 10 ng/mL basic fibroblast growth factor (FGF-2, Gibco) .

Single cells were then plated on uncoated Petri dishes at a density of 3000 cells/cm2.

The resulting neurospheres were allowed to develop in a 95% air-5% CO₂ humidified atmosphere at 37⁰C.

Six to eight days after plating the neurospheres were collected with a Pasteur pipette and seeded onto poly-D- lysine coated glass cover slips placed into 12-well cell culture plates for calcium imaging experiments or 24- well cell culture plates for immunocytochemistry, and then covered with 1 mL or 500 μL, respectively, of SFM devoid of growth factors.

Example 2 - Neuronal differentiation in SVZ cell cultures with the AMPAkine CX-546 in the range of concentrations 0.8 to 500 micraM. SVZ cells were isolated and differentiated according to Example 1 and prepared for single cell calcium imaging after chronic exposure to 7 days in the presence of CX-546 (0.8 to 500 micraM) and procedures described in "General description of the invention - 3. Single cell calcium imaging".

The evaluation of neuronal calcium responses (Histamine/KCl ratio of responses bellow 0.8) results in a percentage of ranging from 15-45%.

Example 3 - Neuronal differentiation in SVZ cell cultures in absence of AMPAkines or other modulators of ionotropic glutamate receptors. SVZ cells were isolated and differentiated according to Example 1 and prepared for single cell calcium imaging without exposure to the presence of CX-546 or to AMPA and procedures described in "General description of the invention - 3. Single cell calcium imaging". The evaluation of neuronal calcium responses (Histamine/KCl ratio of responses bellow 0.8) results in a percentage of ranging from 5-12%.

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Claims

CIAIMS

1. Process for the treatment of SVZ cells comprising a SVZ cell culture developed in presence of at least an AMPAkine and/or other modulators of ionotropic glutamate receptor.

2. Process, according to the previous claim, wherein the modulators of ionotropic glutamate receptor are AMPA.

3. Process, according to the previous claim, wherein the concentration of the AMPA is at least of lμM.

4. Process, according to claim 1, wherein the AMPAkine is Cx546.

5. Process, according to the previous claim, wherein the concentration of the Cx546 is at least of 5μM.

6. Compositions comprising the SVZ cells treated with the process of any of the previous claims.

7. Compositions, according to the previous claim, further comprising other active substances, preferably active substances with neurological effect.

8. Compositions, according to any of the claims 6 to 7, further comprising pharmaceutically acceptable vehicles, carriers, excipients and/or other substances commonly accepted and used in pharmacology.

9. Compositions, according to any of the claims 6 to 8, in the form of tablets, capsules, ampoules, liquid, gel and/or controlled released formulations.

10. Compositions, according to any of the claims 6 to 9, for use as a medicament.

11. Compositions, according to any of the claims 6 to 9, for use in the treatment or prevention of CNS disorders.

12. Compositions, according to any of the claims 6 to 9, for use as a CNS stimulant.

13. Use of the compositions of any of the claims 6 to 12 to produce a medicament for treating, preventing and/or enhancing CNS conditions.

14. A method for treating, preventing and/or enhancing CNS conditions comprising the administration of an effective amount of a composition described in any of the claims 6 to 12.

15. AMPAkines as inductors of neural stem cells differentiation and/or production of neurons.

16. Compositions comprising AMPAkines to be used in the differentiation of neural stem cells and/or production of neurons.