WO2007003675A2 - Experimental neuroinflammation model, method of obtaining same and applications thereof - Google Patents

Abstract

The invention relates to an experimental neuroinflammation model comprising a transgenic non-human animal, which is characterised in that it expresses, exclusively in astrocytes, a mutated form of calcineurin lacking all or part of the calmodulin binding domain and/or the autoinhibitory domain. Said animal model can be used to study and evaluate the progression of neurodegenerative diseases associated with neuroinflammation and pathological neurological alterations caused by inflammation which lead to neuronal death, as well as to identify anti-inflammatory compounds having an anti-inflammatory effect which is due to astrocytic calcineurin stimulation.

Description

EXPERIMENTAL MODEL OF NEUROINFLAMATION, PROCEDURE OF OBTAINING AND ITS APPLICATIONS

FIELD OF THE INVENTION The present invention relates to animal neuroinflammation models useful for studying and evaluating the progression of neurodegenerative diseases that occur with neuroinflammation and pathological neurological alterations caused by inflammation that lead to neuronal death, as well as to identify anti-inflammatory drugs whose effect Anti-inflammatory is due to a stimulation of astrocyte calcineurin.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases that occur with neuronal death are growing alarmingly in developed countries due to various factors, including the gradual aging of the population and changes in lifestyle. There is still no adequate treatment for these diseases.

The study of the pathogenic processes involved in different neurodegenerative diseases as well as in pathological neurological alterations caused by inflammation that lead to neuronal death, such as, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, trauma or sclerosis multiple, it has been shown that they have a common mechanism consisting of inflammation of the nervous tissue.

Neuroinflammation is a complex process that involves cells of the immune system and the central nervous system (CNS), which are intended to repair the damage produced. Inflammation is part of a physiological repair process; However, when this process is not controlled, the inflammatory process spreads, the inflammation loses its repair function and can be the cause of the damage. The inflammatory response in the CNS is thought to favor damage rather than repair function and could explain most CNS pathologies. Therefore, in recent years, numerous therapeutic attempts have been aimed at alleviating it. However, at the moment, it does not appear that any drug has been useful to stop neuroinflammation. There seems to be a physiological system to defend against aggressions that lead to dysfunction and neuronal death. In a previous study it has been seen that insulin-like growth factor type I (IGF-I) is able to modulate in vitro the astrocyte inflammatory response to certain cytokines, in a process that is inhibitory by cyclosporin A, which suggests that it does so through calcineurin stimulation (Pons S, Torres-German I. Insulin-like growth factor-I stimulates dephosphorylation of ikappa B through the serme phosphatase calcineurin (protein phosphatase 2B). J. Biol.Chem. 2000; 275 (49): 38620-5). Therefore, a possible therapeutic approach to the processes that occur with neuronal dysfunction and death is to enhance this defensive system through drugs that stimulate it. One way to do this is to use derivatives of endogenous protective substances as drugs. In this sense, neuroprotective factors such as NGF neurotrophins (nerve growth factor), BDNF (neurotrophic factor have been used

- derived from the brain) or CNTF (ciliary neurotrophic factor) among others. However, the use of these substances poses difficulties of various kinds, from the proper way of administering them to reach the diseased nervous tissue to avoid unwanted side effects. An alternative strategy is to use drugs that modulate the molecular pathways involved in the neuroprotective actions of these endogenous substances. It has now been found, surprisingly, that a phosphatase, calcineurin, seems to be involved in the process of protection against neuroinflammation, contrary to what was believed since traditionally that phosphatase inhibition has been used, for example, with cyclosporine A, as a therapeutic means to curb inflammatory processes in tissues outside the nervous system. This finding allows to develop a new procedure for the identification of therapeutic compounds for neurodegenerative diseases that occur with neuroinflammation and / or for the treatment of neurological pathological alterations caused by inflammation that lead to neuronal death, based on the use of compounds with calcineurin activity or that activate calcineurin to stop neuronal death from neuroinflammation. Such compounds can constitute an adjuvant therapy in the treatment with neuroprotective drugs in general since they facilitate their action by curbing the pathogenic process. Calcineurin is one of the main calmodulin binding proteins in the brain and plays a critical role in the coupling of Ca ²⁺ signals to cellular responses as well as in the activation of T cells. Its calmodulin stimulation ensures coordinated regulation of its phosphatase activity under the control of Ca " ⁺ / calmodulin. The calcineurin dephosphorylates numerous phosphoproteins, including, histones, the myosin light chain and the regulatory sub-unit of the cAMP-dependent kinase. The calcineurin is a heterodimer composed of calcineurin A, a catalytic sub-unit of approximately 60 kDa and by calcinerurin B, a sub-unit of approximately 19 kDa regulating the binding of Ca ²⁺ For a review on calcineurin, its structure, functional organization of domains , calcium regulation and functions can be seen, for example, Klee et al., Minireview: "Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin ", 1998, J. Biol. Chem. 273 (2): 13367-13370.

Calcineurin plays a role in the inhibition of neuronal death by glutamate-mediated neurotoxicity (US 6362160) as well as in the decrease in phosphorylation of the tau protein involved in Alzheimer's Disease (US 6444870).

SUMMARY OF THE INVENTION It has now been found, surprisingly, that calcineurin overexpression in astrocytes, both in primary cultures of such glial cells and in transgenic mice, protects neurons from death induced by inflammatory agents. Therefore, calcineurin constitutes a new therapeutic target for the treatment of neurodegenerative diseases that occur with neuroinflammation and / or for the treatment of neurological pathological alterations caused by inflammation that lead to neuronal death, in particular, for the treatment of the inflammatory phase of said diseases and pathological alterations, which allows establishing a new experimental model for the screening of compounds potentially useful for the treatment of said diseases and pathological alterations. Therefore, in one aspect, the invention relates to a method for the identification of anti-inflammatory compounds whose anti-inflammatory effect is based on the activation of astrocyte calcineurin to stop neuronal death by neuroinflammation. In a particular embodiment, two steps of identification of such compounds, one, in vitro, as a high performance system for initial screening in cells of the nervous system, and another, in vivo, using experimental model animals that express, exclusively in astrocytes, an active variant of calcineurin, either adjustable or non-adjustable form. Gene constructs containing the coding sequence of said active calcineurin variant under the control of a promoter, such as a functional promoter in astrocytes, plasmids or vectors containing said gene constructs as well as astrocyte cultures transfected with said vectors constitute a Additional aspect of this invention. Such transfected astrocyte cultures can be used in a procedure for in vitro identification of anti-inflammatory compounds whose anti-inflammatory effect is based on the activation of astrocyte calcineurin to stop neuronal death by neuroinflammation.

In another aspect, the invention relates to transgenic non-human animals that express, exclusively in astrocytes, an active variant of calcineurin, either in a regulable or non-regulable manner; said non-human animals are useful for identifying in vivo anti-inflammatory compounds whose anti-inflammatory effect is based on the activation of astrocyte calcineurin, whereby neuronal death can be stopped by neuroinflammation. Such transgenic non-human animals constitute an additional aspect of this invention. In a particular embodiment, said transgenic non-human animals express the astrocyte calcineurin in an adjustable (inducible) manner and have been obtained by crossing two parental transgenic lines incorporating the astrocyte calcineurin expression regulating elements. Some of the parental transgenic lines used to obtain said transgenic non-human animals constitute additional aspects of the present invention.

In another aspect, the invention relates to the use of said transgenic non-human animals that express, exclusively in astrocytes, an active variant of calcineurin, to study the etiopathogenic mechanisms of neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation. that lead to neuronal death, or to identify neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to assess the course or progression of diseases neurodegeneratives that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to identify and evaluate potentially therapeutic compounds against neurodegenerative diseases that course with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to identify anti-inflammatory compounds that act by stimulating astrocyte calcineurin.

In another aspect, the invention relates to a method for the identification of a potentially useful compound to block neuronal death by inflammation, based on its ability to stimulate astrocyte calcineurin.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an antagonistic compound of neuronal death processes by inflammation selected from calcineurin and an astrocyte calcineurin activator, and, optionally, a pharmaceutically acceptable excipient.

In another aspect, the invention relates to the use of calcineurin or an astrocyte calcineurin activator in the development of a pharmaceutical composition for the treatment or prophylaxis of neurodegenerative diseases that occur with neuroinflammation and / or neurological pathological alterations caused in mammals, preferably in humans, by inflammation that lead to neuronal death.

In another aspect, the invention relates to a vector comprising the nucleotide sequence encoding a dominant non-functional mutated form of astrocyte calcineurin under the control of a promoter. In another aspect, the invention relates to a vector comprising calcineurin or an astrocyte calcineurin activator.

In another aspect, the invention relates to a method for obtaining a non-human animal model of a neurodegenerative disease comprising crossing said transgenic non-human animal provided by this invention, with an animal of the same species, either wild type (wt) or well genetically manipulated with genes involved in neurodegenerative diseases, and separate useful animals as animal models of neurodegenerative diseases. The nonhuman animals obtainable according to said method constitute an additional aspect of this invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the result of illustrative immunoblots that overexpression of calcineurin in astrocytes nullifies the expression of inflammatory molecules. After an inflammatory stimulus with tumor necrosis factor alpha (TNFα) (10 ng / ml) or with bacterial lipopolysaccharide (LPS) [1 μg / ml) [Example 1], astrocyte calcineurin is able to inhibit the expression of molecules involved in the inflammatory cascade such as cyclooxygenase 2 (COX-2, panel A) and inducible nitric oxide synthetase (ÍNOS2, panel B). The lower blots show the content of β actin to indicate the same protein load per lane. Figure 2 consists of illustrative bar diagrams that the anti-inflammatory response after overexpression of astrocyte calcineurin is mediated by the inhibition of transcription factors NFkB (nuclear factor kappa B (NFkB) and NFAT (nuclear cell factor T activated) To determine the activity of two transcription factors involved in inflammation mechanisms, such as NFkB and NFAT, astrocyte cultures transfected with the vector identified as pΔCaN in this description (Example 1), were co-transfected with reporter systems for NFkB (panel A) or NFAT (panel B). Such report systems consist of plasmids that carry 3 'NFkB or NFAT activation DNA sequences (pNFkB or pNFAT) and a secretable alkaline phosphatase (SEAP) sequence with that the activation of said transcription factors is measured by the luminescence of the medium-secreted alkaline phosphatase (SEAP reporter system, Roche). s control carry empty vectors pCMV (Clontech). After stimulation with the inflammatory agents LPS or TNFα (data not shown), an inhibition of the stimulation of said transcription factors was observed in astrocytes co-transfected with pΔCaN and pNFkB (pΔCaN + pNFkB) or with pΔCaN and pNFAT (pΔCaN + pNFAT) Astrocytes expressing pΔCaN do not show detectable levels of transcription factor activity after LPS stimulation. These results demonstrate that calcineurin inflammation inhibition includes inhibition of NFkB and NFAT activity. Figure 3 consists of illustrative bar diagrams that astrocyte calcineurin stops neuronal death by apoptosis after inflammatory stimuli Co-cultures of neurons and astrocytes transfected with pΔCaN or with the empty vector (pCMV) were stimulated with TNFα (panel A) or with LPS (panel B), and, to At the indicated times, neuronal apoptosis was measured by immunofluorescence for active caspase-3 (CeIl Signaling 1: 500). Cultures were contracted to determine neurons with the anti-β-tubulin III antibody (1: 3000, Promega). The results show that calcineurin expression in astrocytes is sufficient to stop neuronal death by apoptosis evaluated by lowering caspase-3 levels in the cell nucleus with confocal microscopy.

Figure 4 consists of illustrative bar diagrams that astrocyte calcineurin protects neurons from the toxic accumulation of reactive oxygen species (ROS) generated in inflammation. ROS was determined in co-cultures of neurons-astrocytes transfected with pΔCaN by adding dichlorofluorescein diacetate, after inducing inflammation either with LPS (panel A) or with TNFα (panel B). Control cultures contained the empty vector (pCMV). It can be seen that astrocyte cultures alone do not produce ROS in response to pro-inflammatory stimuli (LPS or TNFα). Figure 5 is an explanatory diagram of the Tet-off system (Clontech) for inducible expression of calcineurin in astrocytes. A first transgenic construct is generated containing the murine acid fibrillar glial protein promoter (mGFAP) specific for astrocytes that directs the expression of the artificial bacterial protein tTA. This construction is introduced by conventional transgenesis techniques in non-human animals, for example, mice, generating the I ^{to the} transgenic line of non-human animals. 2 ^to transgenic line of non - human animals, eg, mice, is generated by using a construct containing a sensitive site tTA (TRE) located immediately before the promoter (P) expression in mammalian cells cytomegalovirus (CMV) which, when activated by TRE, directs the expression of a mutated (variant) form of calcineurin (ΔCaN) that has the property of being constitutively active. Once these two independent transgenic lines are obtained, they cross each other (X) to obtain a doubly transgenic line that has the property of expressing said ΔCaN only in astrocytes, only when the animal has active tTA. This protein is inactivated by the administration in the drinking water of doxycycline (Dox), a tetracycline analog.

Figure 6 consists of illustrative bar diagrams that astrocyte calcineurin reduces the level of inflammatory proteins that are produced in the cerebral cortex after an inflammatory stimulus. In double transgenic animals (AIC), which inductively express an active variant of calcineurin (ΔCaN) in astrocytes, the expression of COX-2 and ÍNOS2 is greatly reduced after stimulation with the inflammatory agent LPS in the cerebral cortex. The AIC ± DOX animals were administered LPS (0.5 μg / ml) by stereotaxy at the coordinates (anteroposterior: - 1.3 mm; ventral: 3 mm and lateral: -1.5 mm). After 3 days, the animals were sacrificed under anesthesia (0.5 g / kg tribromoethanol). Blocks (1 mm ³ ) of cerebral cortex around the injection site were homogenized and a westera blot was performed, as described in Example 1, to determine the content in the perilesion zone of COX-2 (panel A) and ÍNOS2 (panel B). The in vivo results show that calcineurin reduces the production of inflammatory molecules (COX-2 and ÍNOS2).

Figure 7 consists of illustrative photographs that overexpression of astrocyte calcineurin results in a reduction of the glial reaction in the area surrounding the lesion. In AIC mice a decrease in the glial reaction that occurs around a traumatic lesion was observed, unlike AIC + Dox animals that have calcineurin expression blocked by the administration of doxycycline (Dox). The glial reaction was evaluated by immunofluorescence with an anti-MHCU antibody that detects reactive microglia.

DETAILED DESCRIPTION OF THE INVENTION

1. Calcineurin: Therapeutic Diana

The invention is based on the identification and exploitation of a new therapeutic target for the treatment of neurodegenerative diseases that occur with neuroinflammation and pathological neurological alterations caused by inflammation that lead to neuronal death, which allows establishing a new experimental model for the study of said pathologies and for screening (identification and / or selection) of compounds potentially useful for the treatment of said diseases and neurological pathological alterations.

As used herein, the term "neurodegenerative disease" refers to a disease associated with a progressive and specific loss of neurons (neuronal death), for example, Parkinson's disease, Huntington's disease, Alzheimer's disease, inflammatory brain disease, multiple sclerosis, amyotrophic lateral sclerosis, dementia associated with immunodeficiency virus human (HIV), etc. As is known, the process of neuronal death causes, at some point in its development, a local inflammatory reaction, so stopping the inflammation, the evolution of the disease is delayed or stopped.

The term "neuroinflammation," as used herein, is a general term that describes the characteristic changes that occur in the brain or spinal cord in response to autoimmune, infectious, ischemic or traumatic damage. In general, neuroinflammation is characterized by an activation and / or proliferation of glial cells at the site of injury.

Likewise, the term "neurodegenerative diseases that occur with neuroinflammation" includes neurodegenerative diseases in which the neurodegenerative process includes a neuroinflammatory process that may be the initial cause of neuronal death or that may be associated with the initial cause of neuronal death; These two possibilities have not been able to be distinguished with certainty except in the case of Multiple Sclerosis where there is strong evidence that the inflammatory process is what initiates neuronal death. Illustrative, non-limiting examples of neurodegenerative diseases with neuroinflammation include Alzheimer's disease, Parkinson's disease, Huntington's disease, inflammatory brain disease, multiple sclerosis, amyotrophic lateral sclerosis, dementia associated with the virus human immunodeficiency (HIV), etc. The term "pathological neurological alterations caused by inflammation leading to neuronal death" includes any neurological alteration, caused by inflammation, which results in neuronal death. Illustrative non-limiting examples of such pathological neurological alterations caused by inflammation leading to neuronal death include dementia caused by the human immunodeficiency virus (HIV) causing acquired immunodeficiency syndrome (AIDS), traumatic brain and spinal injuries, cerebral infarction or ischemia, prion diseases, for example, Creutzfeld-Jacob disease, etc.

The new therapeutic target identified in the present invention is the calcineurin enzyme, a phosphatase, whose role in the inhibition of neuroinflammation was not known. In fact, traditionally, calcineurin has been considered as a mediator of pro-inflammatory processes. In a particular embodiment, the new experimental model provided by this invention for the screening of compounds potentially useful for the treatment of neurodegenerative diseases that occur with neuroinflammation and pathological neurological alterations caused by inflammation leading to neuronal death, based on calcineurin, comprises an in vitro phase as a high performance system for initial screening of potentially useful compounds in astrocytes , and an in vivo phase, for pre-clinical validation of the selected compounds, using experimental model animals that express, exclusively in astrocytes, an active variant of calcineurin either in a regulable or non-regulable way.

It is known that activated glia (astrocytes and microglia) contributes to neuronal damage, via the release of proinflammatory and neurotoxic factors. Such factors include proinflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interleukin 1 (IL-I), nitrogen reactive species, proteases, reactive oxygen species (ROS), eicosanoids and excitatory amino acids (Gao HM, Liu B, Zhang W and Hong JS. Novel anti-inflammatory therapy for Parkinson's disease. Trends Pharmacol. Sci. 2003; 24: 395-401).

It has now been observed that calcineurin overexpression in astrocytes, both in primary cultures and in transgenic mice, protects neurons from death induced by inflammatory agents. Specifically, in the presence of an infection or inflammatory stimulus, calcineurin is able to inhibit the cascade of the inflammatory reaction by preventing translocation to the cell nucleus of transcription factors involved in the inflammatory reaction, such as the NFkB and NFAT factors. This blockage results in the inhibition of the release of inflammatory signals or cytokines in the neuronal environment, thus protecting neurons from death due to inflammation. Therefore, different neurodegenerative diseases that occur with neuroinflammation, as well as pathological neurological alterations caused by inflammation that lead to neuronal death, may be subject to. treatment with calcineurin and / or calcineurin activators in order to stop neuronal death due to inflammation.

In the present invention a model of death by inflammation has been used in which two inflammatory agents are used interchangeably, a pro-inflammatory cytokine (TNFα) or a bacterial toxin (LPS) (Alian SM & Rothwell NJ. Cytokines and acute neurodegeneration. Nature Rev. Neurosci. 2001; 2: 734-44; Kim Y, Moon JS, Lee KS, Park SY, Cheong J, Kang HS, Lee HY, and Kimb HD. Ca2 + / calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NFKB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells. Biochem Biophys Res. Commun. 2004; 314: 695-703).

In an experiment of the present invention, said inflammatory agents (TNFα and LPS) were administered to rat primary astrocyte cultures previously transfected with a plasmid, called pΔCaN in this description (Example 1), which comprises, under the control of the minimum promoter CMV mammalian cell expression, a nucleic acid sequence encoding an active variant of truncated calcineurin that lacks the calmodulin and autoinhibitory binding domains comprising amino acid 1 to 390 of the amino acid sequence of calcineurin A, A isoform. Before an inflammatory stimulus, said active variant of calcineurin expressed by transfected astrocytes is capable of inhibiting the expression of molecules involved in the inflammatory cascade such as COX-2 and ÍNOS2 (Example 1, Figure 1). In another experiment of the present invention, it was observed that the inhibition of the inflammatory response exerted by calcineurin was mediated through the inhibition of inflammatory cascade transcription factors, such as NFkB and NFAT. To do this _^ cultures of rat astrocytes transfected with said plasmid pΔCaN were co-transfected with reporter systems for NFkB or for NFAT which consisted of plasmids carrying 3 'sequences of NFkB or NFAT activation DNA, called pNFkB and pNFAT, respectively, and a nucleotide sequence encoding the secreted form of the alkaline phosphatase as a reporter system (SEAP reporter system, Roche) whereby the activation of said transcription factors can be measured by the luminescence of the alkaline phosphatase secreted into the medium. After stimulation with the inflammatory agents LPS or TNFα, an inhibition of said transcription factors was observed. These results show that, in cultures of astrocytes co-transfected with calcineurin and with reporter systems for NFkB or NFAT, there was an inhibition of these transcription factors when stimulated with the inflammatory agents LPS or TNFα unlike controls (Figure 2).

In another experiment of the present invention, it has been shown that astrocyte calcineurin was able to stop neuronal death after an inflammatory stimulus in astrocyte-neuron co-cultures. Briefly, such co-crops are they transfected with pΔCaN and, after stimulation with TNFα or LPS at different times (Figure 3), neuronal apoptosis was measured by immunofluorescence with an antibody against active caspase-3 (CeIl Signaling 1: 500). Cultures were contracted to determine neurons with the anti-β-tubulin III antibody (1: 3000, Promega). The results showed that calcineurin expression in astrocytes was able to stop neuronal death by apoptosis evaluated by the decrease of caspase-3 levels in the cell nucleus (confocal microscopy) at 3 hours (TNFα) or at 15 hours (LPS) of the proinflammatory cascade initiated (Figure 3). - Likewise, in another experiment of the present invention it was revealed that overexpression of calcineurin in astrocytes protected neurons from the accumulation of ROS, another marker of neuronal death by inflammatory mechanisms. In this particular case, the determination of ROS was done in co-cultures of astrocytes-neurons by adding a compound, dichlorofluorescein diacetate (DCFDA) which, in the presence of intracellular hydrogen peroxide, oxidizes into a fluorescent compound, dichlorofluorescein (Example 2, Figure 4).

In order to carry out the aforementioned in vitro experiments, a vector was previously obtained, the so-called pΔCaN in this description (Example 1), which comprises the nucleotide sequence encoding an active variant of calcineurin under the control of a CMV promoter. Said vector was used to transfect astrocytes.

Additionally, for the performance of in vivo experiments, transgenic non-human animals (mice) were generated that expressed a permanently active form of calcineurin only in astrocytes. Such transgenic non-human animals can be used, for example, as experimental models to identify in vivo anti-inflammatory compounds whose anti-inflammatory effect is based on the activation of astrocyte calcineurin in order to stop neuronal death by neuroinflammation, and constitute an aspect Additional of this invention. Likewise, these animals can be used, among other applications, to study and identify human neurodegenerative diseases that occur with inflammatory processes in which calcineurin intervenes or pathological neurological alterations caused by inflammatory processes in which calcineurin intervenes leading to neuronal death . 2. Transgenic non-human animal of the invention

In another aspect, the invention relates to a transgenic non-human animal, hereinafter, a transgenic non-human animal of the invention, characterized in that it expresses, exclusively in astrocytes, an active variant of calcineurin. Calcineurin is a heterodimer composed of a catalytic sub-unit

(calcineurin A) and a regulatory sub-unit of the Ca junction (calcineurin B) [Klee et al., Minireview: "Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin", 1998, J. Biol. Chem. 273 ( 2): 13367-13370]. The calcineurin A catalytic sub-unit has a catalytic domain, a calcineurin B binding domain, a calmodulin binding domain (Ca ⁺ binding) and an adjacent area comprising the autoinhibitory domain. Three isoforms of the calcineurin A sub-unit whose amino acid sequences are widely conserved in different organisms have been described. The location of the domains present in calcineurin A varies slightly depending on the isoform and the organism. In a particular embodiment, said 4 domains present in the calcineurin A catalytic sub-unit comprise amino acids 1-327 (catalytic domain), amino acids 328-390 (calcineurin B binding domain), amino acids 391-414 (a binding domain calmodulin (binding of Ca ²⁺ )) and amino acids 415-521 (adjacent area comprising the auto-inhibitory domain (amino acids 467-491)). As is known, calmodulin activates calcineurin because it inhibits the action of the auto-inhibitory domain, therefore, any mutation (insertion, deletion or substitution) that prevents the binding of calmodulin to its binding domain and / or that inhibits the action of the autoinhibitory domain, for example, the total or partial deletion of said calmodulin binding domain and / or said autoinhibitory domain, results in a permanent active calcineurin variant as it is not regulable.

As used in this description, the term "active variant of calcineurin" refers to any mutated form of calcineurin (mutant calcineurin) that is not regulable. maintains the phosphatase activity of calcineurin. By way of illustration, not limitation, said active variant of calcineurin can be a mutated form of calcineurin that lacks all or part of the calmodulin binding domain and / or the auto-inhibitory domain, such that said domains are not functional and Therefore, they cannot regulate calcineurin, which becomes permanently active. In a particular embodiment, said active calcineurin variant it is a mutated form of the calcineurin A sub-unit that lacks the calmodulin binding domain and the adjacent autoinhibitory zone (which comprises the autoinhibitoral domain), such as a calcineurin variant A isoform Aa, brain specific, truncated in the domains of calmodulin and autoinhibitory binding, obtained by deletion of said domains, comprising amino acid 1 to 390 of the amino acid sequence of said native calcineurin A (Klee et al., Minireview: "Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin ", 1998, J. Biol. Chem. 273 (2): 13367-13370). Said calcineurin variant is a permanently active variant because it is not regulable and preserves the phosphatase activity of native calcineurin. Alternatively, active variants of calcineurin can be obtained by introducing additional amino acids into said calmodulin and / or autoinhibitoiro binding domains or by replacing some amino acids with others so that said imitating domains are not functional.

As used in this description, the term "non-human animal" includes any non-human animal, among which are non-human mammals, such as non-human primates or rodents, for example, rats or mice. In a particular embodiment, said transgenic non-human animals are transgenic mice.

Said non-human animal may have virtually any known genetic background, that is, it may be a wild-type animal (wt) or a genetically engineered animal, for example, a transgenic, mutant or deficient animal (KO).

According to the invention, overexpression of the active variant of calcineurin occurs exclusively in astrocytes. In a particular embodiment, the expression of said active variant of calcineurin in astrocytes is controlled by a transcription regulatory sequence comprising a specific promoter of said cells and an inducible system of expression of said active variant of calcineurin. By way of illustration, not limitation, in a specific embodiment, said astrocyte specific promoter is the glial fibrillar acid protein (GFAP) promoter. The use of specific astrocyte promoters has the advantage that the active calcineurin variant will only be expressed in the astrocytes of the transgenic non-human animal of the invention. In vivo expression of astrocyte calcineurin can be carried out in a regulated or unregulated manner, for example, by the use of inducible protein expression systems. Virtually any inducible protein expression system can be used in the present invention, for example, the tet-off, tet-on, Cre-lox, Cre-tet, etc .; however, in a particular embodiment, said inducible system system of the expression of said active calcineurin variant is the tet-off system (Clontech).

The transgenic non-human animal of the invention can be obtained by conventional methods of transgenesis that allow overexpression of an active variant of calcineurin exclusively in astrocytes. Information on transgenesis methods can be found, for example, in "Mouse genetics and transgenics: A practical approach", Jackson & Abbot, 1999, Oxford University Press (http: // www .oup.co.uk); "Mouse Genetics: Concepts and Applications", Silver, 1999, (http://www.informatics.jax.org/silver/contents.shtml); and "Gene targeting: A practical approach", Joyner, 1999, in The Practical Approach Series, ed. B.D. Hames Oxford University Press (http://www.oup.co.uk).

As used herein, the term "transgenesis process" refers to any technique or procedure that allows the integration into, at least, a series of cells of a living organism of an exogenous gene, or "transgene", and which confers to these cells and to the organism that carries them a new biological property. Said transgene or exogenous gene refers to a DNA that is not normally resident, nor present in the cell to be transformed; in this case, said transgene includes the DNA sequence encoding an active variant of calcienurin.

In a particular embodiment, said transgenesis process that leads to the expression of an active variant of calcineurin exclusively in astrocytes of the transgenic non-human animal of the invention, is based on a conventional transgenesis process performed in the embryonic phase of said animal of such so that the future astrocytes of the animal express the active variant of calcineurin. The production of these transgenic animals can be carried out by a person skilled in the art based on existing knowledge in the state of the art on transgenic animals (Bedell MA, Jenkins NA, Copeland NG. Mouse models of human disease. Part I: techniques and resources for genetic analysis in mice Genes Dev. 1997 Jan 1; 11 (1): 1- 10. Bedell MA, Longsword DA, Jenkins NA, Copeland NG. Mouse models of human disease Part II: recent progress and future directions. Genes Dev. 1997 Jan 1; 11 (1): 11-43).

In another particular embodiment, said transgenesis process that leads to the expression of an active variant of calcineurin comprises the transformation of astrocytes of a fully developed non-human animal, so as to express said active variant of calcineurin. This objective can be achieved by the introduction into astrocytes of a vector comprising a nucleic acid sequence encoding an active variant of astrocyte calcineurin under the control of a specific astrocyte promoter, in order to transform said astrocytes to express said form. dominant non-functional calcineurin mutant. Said vector can be a viral vector or a non-viral vector, preferably a viral vector since transgenesis with viral vectors has the advantage of being able to direct the expression of a foreign gene in adult tissues with relative precision. The introduction of said vector into astrocytes of the non-human animal to be transformed can be carried out by any conventional method. Transgenesis with viral vectors allows expression in astrocytes near the injection site of the viral vector, but not globally in all astrocytes (as is the case of transgenic non-human animals obtained by conventional methods of embryonic phase transgenesis, as previously described). The expression of the active calcineurin variant in the astrocytes of the transgenic non-human animal of the invention can be regulated or unregulated, preferably regulated. Therefore, in a particular embodiment, the transgenic non-human animal of the invention is obtained by a conventional transgenesis process in which overexpression of the active calcineurin variant can be regulated by an appropriate system, which allows for better control and use of the animal Illustrative examples of such transgenesis processes that allow for regulated expression of the transgene involve the use of conventional systems, such as tet-off, tet-on, Cre-tet, Cre-lox, etc. systems, which are known by the subject matter experts. By way of illustration, not limitation, transgenic non-human animals with adjustable expression of the active calcineurin variant can be obtained by using the tet-off system (Rennel & Gerwins. (2002) How to make tetracycline-regulated transgene expression go on and off Anal Biochem. 309 (l): 79-84; Schonig & Bujard. (2003) Generating conditional mouse mutants via tetracycline-controlled gene expression. In: Transgenic Mouse Methods and Protocols, Hofker, M, van Deursen, J (eds.) Humana Press, Totowa, New Jersey, pp. 69-104).

In a particular embodiment, the transgenic non-human animals provided by this invention are transgenic mice that express an active variant of calcineurin only in astrocytes and, in addition, in a regulable manner, by employing an inducible system of in vivo expression of said active variant of calcineurin, such as the tet-off system, the tet-on system, the Cre-lox system or the Cre-tet system, preferably the tet-off system. In a particular embodiment, the transgenic non-human animals provided by this invention are transgenic mice that express an active variant of calcineurin only in astrocytes and, in addition, • in an adjustable manner, by using the inducible system for protein expression based on the tet-off system (Clontech). To this end, a first transgenic mouse was developed for one country whose genome comprises the murine GFAP promoter that controls the expression of the tetracycline transactivator (tTA) (a protein that activates the TRE site in the other transgene of the tet-off system ). On the other hand, a second transgenic mouse was generated whose genome comprises the nucleotide sequence encoding an active variant of calcineurin (ΔCaN) under the control of the minimum expression promoter in CMV mammalian cells and, in position prior to said coding sequence of said active variant of calcineurin, the nucleotide sequence corresponding to the tTA protein response element (TRE) in order to make it dependent on this protein. The mouse resulting from the crossing of said first and second transgenic mice is a transgenic animal, called the AIC mouse in this description, which expresses an active variant of calcineurin exclusively in astrocytes in a regulable and tTA-dependent manner. The inducible system of the expression of said active variant of calcineurin in astrocytes ceases to produce it when the animals are administered a tTA analogue, such as doxycycline (Dox). The procedure for obtaining said AIC mouse is shown schematically in Figure 5.

Therefore, in a particular embodiment, the genome of the transgenic non-human animal of the invention comprises, under the control of the GFAP promoter, the nucleotide sequence encoding an active variant of calcineurin operably linked to an inducible system of expression of said active variant of calcineurin selected from the tet-off system, the tet-on system, the Cre-lox system and the Cre-tet system, preferably the tet-off system.

A first in vivo experiment of the present invention showed that, in AIC mice (expressing an active variant of calcineurin), the expression of COX-2 and ÍNOS2 is greatly reduced after causing inflammation with LPS in the cerebral cortex ( Figure 6). Briefly, AIC mice and their controls (AIC + Dox) were administered LPS by stereotaxy at certain coordinates. After 3 days, the animals were sacrificed under anesthesia and pieces of cerebral cortex were removed around the injection site that were homogenized and a western blot was performed, as described in Example 1 to determine the content in the perilesion zone of COX-2 and ÍNOS2. The results obtained in vivo demonstrate that calcineurin reduces the production of inflammatory molecules (COX-2 and ÍNOS2).

In another in vivo experiment of the present invention a penetrating lesion (situation similar to what occurs in brain trauma) was performed on the animals. The AIC mice showed a decrease in the glial reaction around the lesion unlike animals that have calcineurin expression blocked (for example, by administration of doxycycline). The evaluation of the tissue reaction was performed by immunofluorescence with an anti-MHCII antibody that detects reactive microglia (Example 3). The results obtained are shown in Figure 7 and show that overexpression of astrocyte calcineurin results in a reduction of the glial reaction in the area surrounding the lesion.

These two in vivo experiments show that calcineurin activation is sufficient to block inflammation-related damage, and, consequently, both calcineurin and an astrocyte calcineurin activator can be used in the treatment or prophylaxis of pathological neurological processes that they study with neuroinflammation, for example, neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused in mammals, preferably in humans, by inflammation that lead to neuronal death. The transgenic non-human animal of the invention can also be used, therefore, to study the etiopathogenic mechanisms of neurological pathological processes that occur with neuroinflammation, for example, neurodegenerative diseases that occur with neuroinflammation or neurological alterations. pathological conditions caused by inflammation leading to neuronal death, in particular, those neurodegenerative diseases and pathological neurological alterations in which calcineurin is involved, which constitutes an additional aspect of this invention. In addition, the transgenic non-human animal of the invention can also be used to identify diseases of mammals, preferably human, neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, in particular, those neurodegenerative diseases and pathological neurological alterations in which calcineurin is involved, which constitutes an additional aspect of this invention. In a particular embodiment, the transgenic non-human animal of the invention is used to identify neurodegenerative human diseases that occur with neuroinflammation in which calcineurin is involved. For this, the neurodegenerative process is provoked, for example, Alzheimer's amyloidosis, cerebral ischemia or others in a transgenic non-human animal of the invention, such as the mouse called AIC in this description, and the course of the disease is assessed by determining levels of markers of amyloidosis, ischemia, etc. Neurodegenerative processes that are modulable by the regulated activation of astrocyte calcineurin (in the mouse AIC ± doxycline) will be those where the inflammatory process is important.

In a specific embodiment, to develop the neurodegenerative disease, the transgenic non-human animal of the invention can cross an animal of the same species, either wild type (wt) or genetically manipulated with genes involved in neurodegenerative diseases, for example, APP for Alzheimer's Disease, hungtintin for Hungtinton's Disease, ataxins 1, 2, etc. for ataxias, synuclein or parkin for Parkinson's disease, etc. In the resulting progeny, the impact of neuroinflammation on the progress of the disease in question can be analyzed. This application of the transgenic non-human animal of the invention allows obtaining information on the importance of the inflammatory component in neurodegenerative disease or in the pathological neurological alteration in question.

Therefore, the use of the transgenic non-human animal of the invention to obtain another non-human animal model of a neurodegenerative disease It constitutes an additional aspect of this invention. More specifically, the invention provides a method for obtaining a non-human animal model of a neurodegenerative disease comprising crossing a transgenic non-human animal of the invention with an animal of the same species, either wild type (wt) or genetically manipulated with genes involved in neurodegenerative diseases, for example, APP for Alzheimer's Disease, hungtintin for Hungtinton's Disease, ataxins 1, 2, etc. for ataxias, synuclein or parlone for Parkinson's disease, etc., and separate useful animals as animal models of neurodegenerative diseases. The non-human animal obtainable according to said method constitutes an additional aspect of this invention.

The transgenic non-human animal of the invention can also be used, in addition, to evaluate the course or progression of neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, in particular, those neurodegenerative diseases and alterations. neurological pathologies in which calcineurin is involved, which constitutes an additional aspect of this invention.

Additionally, the transgenic non-human animal of the invention can also be used to study any cell route in which calcineurin is involved, for example, in learning, etc., which constitutes another additional aspect of this invention.

Likewise, as described below, the transgenic non-human animal of the invention can also be used in vivo animal model to (i) identify and evaluate potentially therapeutic compounds against neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation. that lead to neuronal death, in particular, those neurodegenerative diseases and pathological neurological alterations in which calcineurin is involved, and / or to (ii) identify anti-inflammatory compounds that function based on stimulating astrocyte calcineurin. Such applications of the transgenic non-human animal of the invention constitute additional aspects of this invention. 3. Screening of potentially useful compounds in the treatment of neuroinflammation

The transgenic non-human animal of the invention can also be used as an in vivo model to identify anti-inflammatory compounds that function by stimulating astrocyte calcineurin. In this case, if a compound has anti-inflammatory efficacy, it could be determined if it does via calcineurin in astrocytes based on: (i) comparing its effectiveness with that observed in a transgenic non-human animal of the invention, for example, a mouse AIC, (ii) its activity must be inhibitable due to the absence of calcineurin activity, and (iii) if said compound is administered to transgenic non-human animals of the invention (eg, AIC mice), it must not be effective. Consequently, in another aspect, the invention relates to the use of the transgenic non-human animal of the invention in the identification of anti-inflammatory compounds that function based on stimulating astrocyte calcineurin.

In another aspect, the. The invention relates to a method for the identification of a compound potentially useful for blocking neuronal death by inflammation, based on its ability to stimulate astrocyte calcineurin, which comprises analyzing whether a compound has anti-inflammatory activity and determining whether said anti-inflammatory activity Inflammatory is due to a stimulation of astrocyte calcineurin. For the implementation of said procedure, a wild-type biological environment or, advantageously, a biological environment (eg, transgenic) that expresses an active variant of calcineurin or that does not express calcineurin or has its activity canceled, as well as controls can be used respective.

In a particular embodiment, the compounds whose eventual anti-inflammatory activity based on an astrocyte calcineurin stimulation is desired to be tested may have been previously selected in conventional screening systems, for which conventional assays that allow calcineurin phosphatase activity can be used. Illustrative examples of such tests include tests marketed by Calbiochem (Calcineurin Assay Kit and Calcineurin Cellular Activity Assay Kit). Alternatively, the compounds to be tested have not previously undergone a screening and selection process.

In general, to analyze whether a compound has anti-inflammatory activity and determine whether said anti-inflammatory activity is due to an astrocyte calcineurin stimulation, the procedure comprises: on the one hand, contacting the candidate compound with a wild-type biological environment ("wt"), wherein said, "wt" biological environment is a biological environment comprising wild-type astrocytes ("wt"), optionally in the presence of an inflammatory agent, and determine the anti-inflammatory effect of said compound. in said biological environment "wt" by determining the levels of neuronal death markers by inflammatory mechanisms; on the other hand, it contacts the candidate compound with a biological environment "calcineurin control", wherein said biological environment "calcineurin control" is selected from a biological environment "positive calcineurin control", comprising astrocytes expressing a variant active calcineurin, and a biological environment "negative control of calcineurin", comprising astrocytes where calcineurin activity is inhibited, optionally in the presence of an inflammatory agent, and determine the anti-inflammatory effect of said compound in said biological environment " calcineurin control "by determining the levels of neuronal death markers by inflammatory mechanisms; comparing the levels of neuronal death markers of said candidate compound determined in said "wt" biological environment with those obtained in said "calcineurin control" biological environment and / or with those obtained in controls not treated with said compound; and verify that the anti-inflammatory effect of the candidate compound is due to a stimulation of astrocyte calcineurin.

As used herein, the term "biological environment" wt "" refers to a biological environment comprising wild-type astrocytes (wt); In a particular embodiment, said biological environment "wt" is selected from (i) a culture of "wt" astrocytes (ie, expressing astrocyte calcineurin "wt"), (ii) a co-culture comprising astrocytes "wt" and neurons, and (iii) a non-human animal "wt" containing astrocytes "wt". Likewise, the term "biological environment" positive control of calcineurin "", as used herein, refers to a biological environment comprising astrocytes that express an active variant of calcineurin; in a particular embodiment, said "calcineurin positive control" biological environment is selected from (i) a culture of astrocytes expressing an active variant of calcineurin, (ii) a co-culture comprising astrocytes expressing an active variant of calcineurin and neurons, and (iii) a transgenic non-human animal of the invention

The term "biological environment" negative control of calcineurin "", as used herein, refers to a biological environment comprising astrocytes that express a dominant non-functional mutated form of astrocyte calcineurin or that do not express calcineurin, for example, because Astrocyte expression of the calcineurin gene has been suppressed or inhibited (for example, by molecular (genetic) methods such as those based on the expression of siRNA for calceurin, etc., or pharmacological, such as those based on the use of cyclosporin A, etc.), or because they express a mutated form of the astrocyte calcineurin gene encoding a non-functional protein; In a particular embodiment, said "negative calcineurin control" biological environment is selected from (i) a culture of astrocytes that do not express calcineurin, (ii) a co-culture comprising neurons and astrocytes that do not express calcineurin, and (iii) a transgenic non-human animal that does not express calcineurin in astrocytes.

The cancellation of calcineurin activity comprises the transformation of astrocytes so that they express a dominant non-functional mutated form of calcineurin. This objective can be achieved by the introduction into astrocytes of a vector comprising a nucleic acid sequence encoding a dominant non-functional mutated form of astrocyte calcineurin under the control of a promoter in order to transform said astrocytes so as to express said mutated form. non-functional calcineurin dominant. The promoter can be a functional viral promoter in mammalian cells, for example, a cytomegalovirus (CMV) promoter or a specific astrocyte promoter, for example, the glial fibrillar acid protein (GFAP) promoter. Said vector may be a viral vector or a non-viral vector. The transformation of said astrocytes can be carried out by conventional methods known to those skilled in the art.

As used herein, the term "a dominant non-functional mutated form of calcineurin" includes any mutated form of calcineurin that acts as a negative dominant by canceling its biological function. Said dominant non-functional mutated form of calcineurin is expressed by astrocytes as a result of its transformation with a vector comprising the acid sequence. nucleic encoding a dominant non-functional mutated form of astrocyte calcineurin under the control of an appropriate promoter, such as a viral promoter, for example, a potent promoter of type, viral functional in astrocytes, etc. Virtually any dominant non-functional mutated form of calcineurin can be used in order to achieve functional cancellation of the biological activity (biological cancellation) of said enzyme.

Alternatively, the alteration of the biological activity of the function of astrocyte calcineurin may be due to the cancellation of the functional activity of endogenous calcineurin due to the expression of a polynucleotide whose nucleotide sequence encodes an element that inhibits the expression of the gene expression. calcineurin capable of canceling its functional activity. Therefore, in another particular embodiment, the cancellation of the functional activity of endogenous astrocyte calcineurin comprises the transformation of astrocytes by the introduction of a vector comprising a polynucleotide whose nucleotide sequence encodes an element that inhibits calcineurin gene expression. able to cancel its biological or functional activity. As used in the present invention and, as mentioned above, the term "calcineurin gene expression inhibitor capable of nullifying its biological or functional activity" refers to a protein, enzymatic activity or nucleotide sequence, Single or double stranded RNA or DNA, which inhibits the translation of endogenous astrocyte calcineurin into mRNA protein. By way of illustration, said polynucleotide may be: a) a polynucleotide encoding a specific antisense nucleotide sequence of the calcineurin gene or mRNA sequence, b) a polynucleotide encoding a specific calcineurin mRNA ribozyme, c) a polynucleotide encoding a specific calcineurin mRNA aptamer, od) a polynucleotide encoding a small interference RNA (siRNA) specific for calcineurin mRNA. The nucleotide sequences a) -d) mentioned above prevent the expression of the gene in mRNA or mRNA in astrocyte calcineurin, and therefore cancel its biological function, and can be developed by an expert in the field of genetic engineering in function of the existing knowledge in the state of the art about transgenesis and the annulment of gene expression (Clarke, AR (2002) Transgenesis Techniques Principles and Protocols, 2nd Ed Humana Press, Cardiff University;.. Patent US20020128220 Gleave, Martin TRPM-2 antisense therapy;.. Door-Ferandez E et to the . (2003) Ribozymes: recent advances in the development of RNA tools. FEMS Microbiology Reviews 27: 75-97; Kikuchi, et al., 2003. RNA aptamers targeted to domain II of Hepatitis C virus IRES that bind to its apical loop region J. Biochem. 133, 263-270; Reynolds A. et al., 2004. Rational siRNA design for RNA interference. Nature Biotechnology 22 (3): 326-330).

In another particular embodiment, the cancellation of the calcineurin functional activity in astrocytes of the non-human animal is carried out by means of a conventional transgenesis process in the embryonic phase of said animal so that the future astrocytes of said animal are genetically transformed. and lose the ability to produce endogenous astrocyte calcineurin. The development of this type of transgenic animal can be carried out by a person skilled in the art in view of the state of the art on transgenic animals (Bedell MA, Jenkins NA, Copeland NG. Mouse models of human disease. Part I: techniques and resources for genetic analysis in mice Genes Dev. 1997 Jan 1; 11 (1): 1-10 Bedell MA, Longsword DA, Jenkins NA, Copeland NG Mouse models of human disease Part II: recent progress and future directions Genes Dev. 1997 Jan 1; 1 1 (1): 11-43). For the implementation of the previously defined procedure for the identification of potentially useful compounds to block neuronal death by inflammation, based on their ability to stimulate astrocyte calcineurin, the compound to be tested is contacted with said biological environments "wt" and "calcineurin control", optionally in the presence of an inflammatory agent. Therefore, in a particular embodiment, said procedure is carried out in the absence of an inflammatory agent; This way of carrying out said procedure can be used when it is desired to study the possible anti-inflammatory effect of the compound to be tested in situations of traumatic injury to the cerebral cortex in vivo. Alternatively, in another particular embodiment, the previously defined procedure is carried out in the presence of an inflammatory agent. This alternative is suitable for tests in which inflammation is stimulated and the possible anti-inflammatory effect of the compound to be tested either in vitro or in vivo is analyzed. Illustrative, non-limiting examples of inflammatory agents include LPS or TNFα. The levels of inhibition of the inflammatory effects of the test compound in said "wt" and "control" biological environments are evaluated by determining the levels of neuronal death markers by inflammatory mechanisms. Different methods and systems can be used depending on the markers that you want to analyze. In general, these methods and systems depend on the biological environments used.

In a particular embodiment, said biological environment comprises an astrocyte culture (eg, an astrocyte culture expressing astrocyte calcineurin "wt", an astrocyte culture expressing an active variant of calcineurin, or an astrocyte culture with inhibited calcineurin) , in the presence of an inflammatory agent. In this case, the inhibition of the inflammatory effects of the candidate compound to be tested (i.e. its potential anti-inflammatory effect) can be carried out by assessing the expression levels of at least one inflammatory protein, optionally in the presence of a calcineurin inhibitor Practically the expression of any inflammatory protein, both those that are secreted to the environment (eg, cytokines, etc.) and those that are not secreted to the environment (eg, ÍNOS2, etc.), can be used to determine the inhibition of the inflammatory effects of the test compound; by way of illustration, not limitation, the (pro) inflammatory proteins COX-2, INOS2, interleukins (IL), such as IL-I, IL-2, IL-6, IL-12, Rantes, etc., and combinations thereof. However, in a particular embodiment, COX-2 expression levels, ÍNOS2 expression levels or COX-2 and ÍNOS2 expression levels are assessed. The expression levels of said inflammatory protein can be assessed, if desired, in the presence of a calcineurin inhibitor. Therefore, in a particular embodiment, the assessment of the expression levels of said inflammatory protein is carried out in the absence of a calcineurin inhibitor. Alternatively, in another particular embodiment, the assessment of expression levels of said inflammatory protein is carried out in the presence of a calcineurin inhibitor. Virtually any calcineurin inhibitor can be used, for example, cyclosporin A, FK506, etc. In another particular embodiment, said biological environment comprises a co-culture of neurons and astrocytes that express astrocyte calcineurin "wt", a biological environment comprising a co-culture of neurons and astrocytes that express an active variant of calcineurin, or a biological environment comprising a co-culture of neurons and astrocytes that do not express calcineurin (eg, with calcineurin inhibited), in the presence of an inflammatory agent, as previously defined. In this case, the inhibition of the inflammatory effects of the candidate compound to be tested (i.e. its potential anti-inflammatory effect) can be carried out by assessing the survival of the neurons. Virtually any method that allows assessing the survival of neurons can be used. Illustrative, non-limiting examples of such methods for assessing the survival of neurons are based on the determination of caspase-3 levels, ROS, LDH (lactate dehydrogenase), MTT (3-4,5-dimethylthiazol-2-yl -2,5-diphenyltetrazolium bromide), etc., or in the performance of different vital stains, for example, calcein, dichloroflorescein, propidium iodide, etc., and combinations of such methods. In a specific embodiment, the survival of neurons is assessed by determining caspase-3 levels, which can be carried out by immunofluorescence with anti-caspase-3. In another specific embodiment, the survival of the neurons is assessed by determining them. ROS levels, which can be carried out by fluorescence.

In another particular embodiment, said biological environment comprises a non-human animal, such as, for example, a non-human animal ^or wt "containing astrocytes

-. "wt", a transgenic non-human animal of the invention, or a transgenic non-human animal that does not express astrocyte calcineurin, to which an inflammatory agent, as previously defined, has been administered by any appropriate method, for example, by administration of said inflammatory agent directly to the brain of the animal. In this case, the inhibition of the inflammatory effects of the candidate compound to be tested (i.e. its potential anti-inflammatory effect) can be carried out by assessing the expression levels of at least one inflammatory protein, optionally in the presence of a calcineurin inhibitor The inflammatory proteins whose expression can be determined have already been mentioned previously as well as the calcineurin inhibitors optionally present in the evaluation of the levels of expression of said inflammatory proteins. By way of illustration, not limitation, the (pro) inflammatory COX-2, INOS2, 0 interleukin (IL) proteins, such as IL-I, IL-2, IL-6, IL-12, Rantes, etc., may be cited. and combinations thereof. However, in a particular embodiment, COX-2 expression levels, ÍNOS2 expression levels or expression levels of COX-2 and ÍNOS2. Illustrative, non-limiting examples of calcineurin inhibitors include cyclosporine A, FK506, etc.

In another particular embodiment, said biological environment comprises a non-human animal, such as, for example, a "wt" non-human animal containing "wt" astrocytes, a transgenic non-human animal of the invention, or a transgenic non-human animal that does not express astrocyte calcineurin, which has been caused a traumatic injury. Traumatic injury can be located in any part of the brain, for example, in the cerebral cortex (area where most traumatic injuries occur in humans), and can be caused by any conventional method, for example, by a cut, or an abrasion in the area where the traumatic injury will occur. In this case, the inhibition of the inflammatory effects of the candidate compound to be tested (i.e. its potential anti-inflammatory effect) can be carried out by determining the volume of the lesion in the perilesion zone. Virtually any method that allows to determine the volume of the lesion in the perilesion zone can be used to determine the inhibition of the inflammatory effects of the test compound. Illustrative, non-limiting examples of such methods consist in determining the microglial reaction area or the number of dead cells in the perilesion zone, histological stains, for example, with hematoxylin-eosin, fiuorojade, etc. In a particular embodiment, the volume of the lesion in the perilesion zone is established by determining the area of microglial reaction or the number of dead cells.

Once the levels of the corresponding markers of neuronal death have been determined in the "wt" biological environment and in the "calcineurin control" biological environment, their comparison is made between them and against the values obtained in "blanks" or controls that do not they have been in contact with, or to which they have not been administered, the candidate compound to be tested and to select the compounds with anti-inflammatory activity as a previous step to determine if said anti-inflammatory activity is due to an astrocyte calcineurin stimulation. In this sense, the following are selected:. - the candidate compounds that produce levels of neuronal death markers. by smaller inflammatory mechanisms in the biological environment "wt" than in the biological environment "negative calcineurin control"; Candidate compounds that produce levels of neuronal death markers by the same or similar inflammatory mechanisms (ie approximately the same order), in the biological environment "wt" as in the biological environment "positive control of calcineurin" [in this case , if the compound works by stimulating calcineurin, in the "positive calcineurin control" you should not do anything since calcineurin is already active and, therefore, the levels of markers of neuronal death by inflammatory mechanisms are the same or similar in such environments biological "wt" and "positive control of calcineurin"]; and - the candidate compounds that produce levels of neuronal death markers by lower inflammatory mechanisms in the biological environment "wt" than in the "whites".

Subsequently, it is checked whether the anti-inflammatory effect of the selected candidate compound is due to an astrocyte calcineurin stimulation. For this, the determination of the levels of neuronal death markers by inflammatory mechanisms is performed in the presence of an astrocyte calcineurin inhibitor, for example, cyclosporin A, FK506, etc., in a "wt" biological environment and the results obtained. Increased levels of markers of neuronal death by inflammatory mechanisms in the presence ^'of the inhibitor astrocyte calcineurin, relative to a control without inhibitor astrocyte calcineurin, is indicative that the antiinflammatory effect of the compound is due to stimulation of calcineurin astrocitary On the other hand, if the levels of neuronal death markers by inflammatory mechanisms in the presence of the astrocyte calcineurin inhibitor are the same or similar as those obtained in a control without astrocyte calcineurin inhibitor, the anti-inflammatory effect of the compound may be due to a mechanism different from that of astrocyte calcineurin stimulation.

The results obtained with the procedure for identifying potentially useful compounds to block neuronal death due to inflammation, based on their ability to stimulate astrocyte calcineurin, make it possible to select specific anti-inflammatory compounds for the pathology to be treated, in particular, pathological neurological processes that They have neuroinflammation and / or neurodegenerative diseases that present with neuroinflammation and / or pathological neurological alterations caused by inflammation that lead to neuronal death, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, inflammatory brain disease, multiple sclerosis, amyotrophic lateral sclerosis, ischemia or cerebral infarction, HIV-associated dementia, traumatic brain and spinal injuries, heart attack or cerebral ischemia, prion diseases, by. example, Creutzfeld-Jacob disease, etc.

4. Pharmaceutical compositions

The calcineurin or the astrocyte calcineurin activators constitute an adjuvant therapy in the treatment with neuroprotective drugs in general since they facilitate their action by curbing the pathogenic process. Therefore, in another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an antagonistic compound of neuronal death processes by inflammation, such as calcineurin or an astrocyte calcineurin activator, and, optionally, a pharmaceutically acceptable excipient.

The amount of therapeutically effective antagonistic compound of the processes of neuronal death due to inflammation (calcineurin or an astrocyte calcineurin activator) as well as its dosage to treat a pathological state with said antagonistic compound of the processes of neuronal death due to inflammation will depend of numerous factors, among which are the age, the patient's condition, the severity of the disease, the route and frequency of administration, the modulator compound to be used, etc.

The pharmaceutical compositions provided by this invention may be presented in any form of administration deemed suitable for administration by any route, for example, orally, parenterally, rectally, topically, etc., for which pharmaceutically acceptable excipients will be included. necessary for the formulation of the desired administration form. A review of the different pharmaceutical forms of drug administration and of the necessary excipients' to obtain them can be found, for example, in the "Galician Pharmacy Treaty", C Faulí í Trillo, 1993, Luzán 5, SA de Editions, Madrid.

In a particular embodiment, the pharmaceutical composition provided by this invention is a composition intended for use in gene therapy that It comprises a vector, viral or non-viral, and an antagonistic compound of neuronal death processes due to inflammation, such as calcineurin or an astrocyte calcineurin activator. Said vector constitutes an additional aspect of this invention. By way of illustration, not limitation, said vectors may be viral, for example, vectors based on retroviruses, adenoviruses, etc., or non-viral, such as DNA-liposome complexes, DNA-polymer, DNA-polymer-liposome, etc. ["Nonviral Vectors for Gene Therapy", edited by Huang, Humg & Wagner, Academic Press (1999)]. Gene therapy with vectors that provide antagonistic compounds of neuronal death processes due to inflammation, such as calcineurin or astrocyte calcineurin activators in localized neurodegenerative lesions, is a potentially interesting therapy for the treatment of neurodegenerative diseases that occur with neuroinflammation- and / or pathological neurological alterations caused by inflammation that lead to neuronal death, in which calcineurin is involved. In another aspect, the invention relates to the use of calcineurin or an astrocyte calcineurin activator in the elaboration of a pharmaceutical composition for the treatment or prophylaxis of neurodegenerative diseases that occur with neuroinflammation and / or neurological pathological alterations caused in mammals, preferably in humans, by inflammation that lead to neuronal death; Illustrative, non-limiting examples of such diseases and alterations include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, inflammatory brain disease, multiple sclerosis, amyotrophic lateral sclerosis, ischemia or cerebral infarction , HIV-associated dementia, traumatic brain and spinal injuries, cerebral infarction or ischemia, prion diseases, for example, Creutzfeld-Jacob disease, etc. In a particular embodiment, said pharmaceutical composition is a composition intended for use in gene therapy comprising a vector, viral or non-viral, and an antagonistic compound of neuronal death processes by inflammation, such as calcineurin or a calcineurin activator astrocitary The following examples serve to illustrate the invention and should not be considered in a limiting sense of the scope thereof. EXAMPLE 1

Astrocyte calcineurin inhibition of the expression of inflammatory molecules (iNOS and COX-2) caused by inflammatory agents

To determine the anti-inflammatory role of calcineurin in astrocytes, cultures transfected with LPS or with TNFα are stimulated and the anti-inflammatory response is assessed, at a first approximation, by the western blot determination of proteins involved in inflammation, for example, iNOS and COX-2.

Briefly, primary astrocyte cultures of 3-4 days old rat cerebral cortex were transfected with the vector identified as pΔCaN obtained from a pCMV vector (Clontech) comprising, under the cytomegalovirus mammalian cell expression promoter (CMV), a DNA sequence that encodes an active variant of calcineurin, specifically, a form of the brain-specific calcineurin A-isoform Aa subset, truncated in the calmodulin-binding and auto-inhibitory domains comprising amino acid 1 to 390 of the amino acid sequence of said calcineurin isoform Aa (hereinafter ΔCaN). Said pΔCaN vector expresses a constitutively active form of calcineurin. The expression of calcineurin is determined by western blotting with a polyclonal anti-calcineurin A antibody that detects the endogenous and truncated forms (active variant of calcineurin). Controls were obtained by transfecting primary astrocyte cultures of rat cerebral cortex (3-4 days of age) with the empty vector pCMV (Clontech). After 1 day, the astrocytes were stimulated with LPS (1 μg / ml) overnight or with TNFα (10 ng / ml) for 30 minutes. A control group received no stimulation. After these times the astrocytes were lysed with 250 μl of lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mg / ml aprotinin, 1 mg / ml leupeptin and florophenylmethylsulfonyl 1 mg / ml). The samples were subjected to immunoblot after electrophoresis and transfer to nitrocellulose membranes. The membranes were incubated with primary anti-COX-2 (Cayman, 1: 1000) and anti-iNOS2 (CeIl Signaling, 1: 1000) antibodies. As a secondary antibody, a peroxidase-labeled rabbit IgG antibody (anti-rabbit-HRP) was used and development was performed with the chemiluminescent compound ECL (Amersham). Bands were analyzed by densitometry (BioRad). The results showed a decrease in the density of the bands when the astrocytes overexpressed calcineurin with respect to the controls. These results demonstrate that astrocyte calcineurin is capable of inhibit the expression of molecules involved in the inflammatory cascade such as COX-2 and iNOS2 (Figure 1).

EXAMPLE 2 Inhibition by astrocyte calcineurin of the accumulation of ROS induced by inflammatory agents in neurons

Astrocyte cultures of rat cerebral cortex transfected with pΔCaN, described in Example 1, are added postnatal rat cerebellum neurons (6-7 days of age). Control cultures were obtained by transfecting primary astrocyte cultures of rat cerebral cortex (3-4 days of age) with the empty vector pCMV (Clontech). The cultures were stimulated with LPS (1 μg / ml) overnight or with TNFα (10 ng / ml) for 30 minutes or with the vehicle (control). After that time the formation of reactive oxygen species (ROS) was measured by adding dichlorofluorescein diacetate (DCFDA) which, in the presence of intracellular hydrogen peroxide, oxidizes and converts to dichlorofluorescein. After the different treatments, the cells were washed with DMEM + 1% FCS (DMEM: Dulbecco modified essential medium; FCS: Fetal calf serum) and incubated with 50 mM DCFDA (10 mM stock solution in dimethylsulfoxide) in DMEM + 1% FCS for 50 minutes at 37 ° C. The cells were washed twice with Krebs buffer at 37 ° C. Finally the cells were solubilized by adding 0.1 N NaOH in 50% methanol and stirred for 10 minutes. The peroxide generation was measured in a FLUOstar fluorescence plate reader (BMG Labtechnologies GmbH, Germany), with excitation lengths of 485 nm, and emission of 520 nm, and a gain of 10. Whites, which were subtracted, they were wells without cells but with DCFDA and processed identically. The results showed that calcineurin overexpression in astrocytes protects neurons from the accumulation of ROS, a marker of neuronal death by inflammatory mechanisms (Figure 4). EXAMPLE 3

Reduction of inflammation in the area surrounding a brain injury due to in vivo overexpression of a truncated calcineurin in astrocytes

3.1 Generation of transgenic mice that overexpress truncated calcineurin (AIC mice)

Transgenic mice, called AIC mice in this description, were generated that overexpress an active variant of calcineurin in astrocytes, by crossing parental transgenic mice carrying two transgenes using the tet-off system (Clontech). For this, the corresponding gene constructs were previously prepared, one of which contained the coding sequence of the tetracycline transactivator (tTA) under the control of the murine acid fibrillar glial protein promoter (mGFAP) and the other containing a tTA-sensitive site ( TRE) located immediately before the minimum cytomegalovirus (CMV) mammalian cell expression operator operatively linked to said nucleotide sequence encoding an active variant of calcineurin identified as ΔCaN in this description (Example 1).

Briefly, I ^to transgenic line was generated by introducing into mice, by conventional methods of transgenesis, a gene construct containing the promoter mGFAP (astrocyte specific) controlling the expression of the tTA. Of the mice that tested positive for the transgene, identified by means of the polymerase chain reaction (PCR), those that best transmitted to the progeny were selected, thereby obtaining I ^{at the} transgenic line.

2 ^to transgenic line was generated by introducing into mice a second DNA construct containing a TRE site responsive to tTA just before the minimal promoter of expression in mammalian cells of CMV operably linked to the nucleotide sequence encoding said variant of calcineurin ( ΔCaN) which has the property of being constitutively active. When activated by TRE, the promoter directs the expression of said ΔCaN (mutated form of calcineurin that has the property of being constitutively active). Mice that tested positive for the transgene (identified by PCR) those that had increased levels of expression of ΔCaN for use as founders 2 transgenic line were selected.

Once these two independent transgenic lines are obtained, the transgenic mice cross each other to obtain the doubly transgenic line (mice AIC) that has the property of expressing ΔCaN only in astrocytes, only when the animal has active tTA. This protein is inactivated by administration to the animal, for example, in the drinking water, of doxycycline (Dox), a tetracycline analog. AIC mice are selected by expression of the transgenes (identified by PCR of their DNA).

3.2 Effect of astrocyte overexpression (in vivo) of a truncated calcineurin on inflammation of the area surrounding a brain injury

AIC mice, which overexpress truncated calcineurin, generated as indicated in section 3.1, were caused a lesion consisting of a cut in the cerebral cortex following the stereotactic coordinates (anteroposterior: -1.3-1.4 mm; ventral: 3 mm and lateral: -1.5 mm) according to the stereotactic atlas of mouse brain (Paxinos G, Watson CR. The mouse brain in stereotaxic coordinates. 3 edn. Sydney 1982). After 3 days, the animals were perfused with 4% paraformaldehyde and sections of the brain were obtained with a vibratome (50 μm) where immunofluorescence was performed. Briefly, the brain sections were washed with PBT [phosphate buffer with 0.1% Triton X-100 and 0.1% BSA (bovine serum albumin)] and incubated with 3% normal goat serum for 30 minutes . After three 10-minute washes with PBT, the sections were incubated with the primary anti-MHCII antibody (Serotec), which identifies the reactive microglia, diluted 1: 1000 in PBT, overnight at 4 ° C. The next day, the cells were washed three times with PBT and incubated for an hour and a half at room temperature with the secondary anti-mouse IgG anti-mouse alexa 488 (obtained in goat) (Molecular Probes). After several washes, the sections were mounted in gelatinized slides, dehydrated and mounted with DEPEX. The quantification of the number of positive cells in the brain sections of animals that express or not calcineurin was performed with a confocal microscope. The results show that AIC mice show a reduction of the inflammatory glial reaction in the perilesion area (Figure 7), which also demonstrates in vivo that astrocyte calcineurin is a neuroinflammation protective agent.

Claims

1. A transgenic non-human animal characterized in that it expresses, exclusively in astrocytes, an active variant of calcineurin.

2. Animal according to claim 1, wherein said active calcineurin variant is a mutated form of calcineurin that lacks all or part of the calmodulin binding domain and / or the auto-inhibitory domain.

3. Animal according to claim 1 or 2, wherein said active calcineurin variant comprises amino acid 1 to amino acid 390 of the amino acid sequence of the calcineurin sub-unit A isoform Aa.

4. Animal according to claim 1, characterized in that it is a non-human mammal, preferably a non-human primate or a rodent, more preferably, a mouse.

5. Animal according to claim 1, wherein the expression of said active variant of calcineurin in astrocytes is controlled by a transcriptional regulatory sequence comprising a specific astrocyte promoter and an inducible system of expression of said active calcineurin variant .

6. Animal according to claim 5, wherein said astrocyte specific promoter is the glial fibrillar acid protein (GFAP) promoter.

7. Animal according to claim 5, wherein said inducible expression system of said calcineurin variant comprises the tet-off system, the busty system, the Cre-lox system or the Cre-tet system.

8. Animal according to claim 1, characterized in that it is a transgenic mouse whose genome comprises, under the control of the GFAP promoter, the nucleotide sequence encoding an active variant of calcineurin operably linked to an inducible system of in vivo expression of said variant active calcineurin selected from the tet-off system, the tet-on system, the Cre-lox system and the Cre-tet system, preferably the tet-off system.

9. Use of a transgenic non-human animal according to any of claims 1 to 8. for:

study the etiopathogenic mechanisms of neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to

- identify . diseases . neurodegeneratives that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to

evaluate the course or progression of neurodegenerative diseases that • occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to

- identify and evaluate potentially therapeutic compounds against neurodegenerative diseases that occur with neuroinflammation or pathological neurological alterations caused by inflammation that lead to neuronal death, or to

- identify anti-inflammatory compounds that act by stimulating astrocyte calcineurin.

10. A procedure for the identification of a potentially useful compound to block neuronal death by inflammation, based on its ability to stimulate astrocyte calcineurin, which comprises

(i) analyze, in a biological environment selected from a biological environment that expresses an active variant of calcineurin and a biological environment that do not express calcineurin or have its activity canceled, if the candidate compound has anti-inflammatory activity, and

(ii) determine whether said anti-inflammatory activity is due to a stimulation of astrocyte calcineurin.

A method according to claim 10, comprising: on the one hand, contacting the candidate compound with a wild type biological environment ("wt"), wherein said "wt" biological environment is a biological environment comprising wild type astrocytes ("wt"), optionally in the presence of an inflammatory agent, and determining the anti-inflammatory effect of said compound in said biological environment "wt" by determining the levels of neuronal death markers by inflammatory mechanisms; - on the other hand, it puts the candidate compound in contact with a biological environment "calcineurin control", where said biological environment. "calcineurin control" is selected from a biological environment "positive calcineurin control", which comprises astrocytes expressing an active variant of calcineurin, and a biological environment "negative calcineurin control", which comprises astrocytes where calcineurin activity is inhibited, optionally in the presence of an inflammatory agent, and determining the anti-inflammatory effect of said compound in said biological environment "calcineurin control" by determining the levels of neuronal death markers by inflammatory mechanisms; - comparing the levels of neuronal death markers of said candidate compound determined in said biological environment "wt" with those obtained in said biological environment "calcineurin control" and / or with those obtained in controls not treated with said compound; and verify that the anti-inflammatory effect of the candidate compound is due to a stimulation of astrocyte calcineurin.

12. The method of claim 11, wherein said "wt" biological environment is selected from (i) a culture of "wt" astrocytes expressing calcineurin astrocyte "wt", (ii) a co-culture comprising "wt" astrocytes expressing astrocyte calcineurin "wt" and neurons, and (iii) a non-human animal "wt" containing astrocyte "wt" astrocytes that express astrocyte calcineurin " wt. "

13. Method according to claim 11, wherein said biological environment

"positive calcineurin control" is selected from (i) a culture of astrocytes expressing an active variant of calcineurin, (ii) a co-culture comprising neurons and astrocytes expressing an active variant of calcineurin, and (iii) an animal Transgenic non-human according to any of claims 1 to 8.

14. The method of claim 11, wherein said "calcineurin negative control" biological environment comprises astrocytes expressing a dominant non-functional mutated form of astrocyte calcineurin or astrocytes that do not express calcineurin, preferably, (i) a culture of astrocytes that do not express calcineurin, (ii) a co-culture comprising neurons and astrocytes that do not express calcineurin, or (iii) a transgenic non-human animal that does not express calcineurin in astrocytes.

15. A method according to claim 1, wherein the anti-inflammatory effect of the candidate compound in a biological environment selected from a biological environment comprising a culture of astrocytes expressing astrocyte calcineurin "wt", a biological environment comprising a culture of astrocytes expressing an active variant of calcineurin, and a biological environment comprising an astrocyte culture with inhibited calcineurin, is determined, in the presence of an inflammatory agent, assessing the expression levels of at least one inflammatory protein, optionally in the presence of a calcineurin inhibitor.

16. The method of claim 11, wherein the anti-inflammatory effect of the candidate compound in a biological environment selected from a biological environment comprising a co-culture of neurons and astrocytes expressing astrocytic calcineurin "wt", a biological environment comprising a co-culture of neurons and astrocytes that express an active variant of calcineurin, and a biological environment comprising a co-culture of neurons and astrocytes with calcineurin inhibited, it is determined, • in the presence of an inflammatory agent, assessing the survival of neurons.

17. The method of claim 11, wherein the anti-inflammatory effect of the candidate compound in a biological environment selected from a non-human "wt" animal containing "wt" astrocytes, a transgenic non-human animal according to any one of claims 1 at 8, and a transgenic non-human animal that does not express astrocyte calcineurin, to which an inflammatory agent has been administered, is determined by assessing the expression levels of at least one inflammatory protein, optionally in the presence of a calcineurin inhibitor .

18. The method of claim 11, wherein the anti-inflammatory effect of the candidate compound in a biological environment selected from a non-human "wt" animal containing "wt" astrocytes, a transgenic non-human animal according to any one of claims 1 to 8 , and a transgenic non-human animal that does not express astrocyte calcineurin, which has been caused a traumatic injury to the brain, is determined by measuring the volume of the lesion in the perilesion zone.

19. A method according to claim 11, wherein the verification of whether the anti-inflammatory effect of the candidate compound is due to an astrocyte calcineurin stimulation is carried out by determining the levels of neuronal death markers by inflammatory mechanisms in the presence of an astrocyte calcineurin inhibitor.

20. A pharmaceutical composition comprising a therapeutically effective amount of an antagonistic compound of neuronal death processes by inflammation selected from calcineurin and an astrocyte calcineurin activator, and, optionally, a pharmaceutically acceptable excipient.

21. Composition according to claim 20, which comprises a vector and an antagonistic compound of the processes of neuronal death by inflammation selected from calcineurin and an astrocyte calcineurin activator.

22. Use of calcineurin or an astrocyte calcineurin activator in the development of a pharmaceutical composition for the treatment or prophylaxis of neurodegenerative diseases that occur with neuroinflammation and / or neurological pathological alterations caused in mammals, preferably in humans, by inflammation that lead to neuronal death

23. Use according to claim 22, wherein said neurodegenerative diseases that occur with neuroinflammation or said pathological neurological alterations caused by inflammation leading to neuronal death include Alzheimer's disease, Parkinson's disease, Huntington's disease, inflammatory disease cerebral, multiple sclerosis, amyotrophic lateral sclerosis, ischemia or cerebral infarction, HIV-associated dementia, traumatic brain and spinal injuries, cranial trauma, craniocerebral trauma, cerebral vascular accident (stroke), heart attack or cerebral ischemia, or Creutzfeld-Jacob disease.

24. A vector comprising the nucleotide sequence encoding an active variant of calcineurin under the control of a promoter.

25. Vector according to claim 24, wherein said active variant of calcineurin is a mutated form of calcineurin that lacks all or part of the calmodulin binding domain and / or the auto-inhibitory domain.

26. A vector according to claim 24 or 25, wherein said active calcineurin variant comprises amino acid 1 to amino acid 390 of the amino acid sequence of the calcineurin sub-unit A isoform Aa.

27. Vector according to claim 24, wherein said promoter is a functional viral promoter in mammalian cells, preferably, a cytomegalovirus (CMV) promoter.

28. Vector according to claim 24, wherein said promoter is a specific astrocyte promoter, preferably, the glial fibrillar acid protein (GFAP) promoter.

29. An astrocyte culture comprising astrocytes transfected with a vector according to any of claims 24 to 28.

30. A co-culture comprising an astrocyte culture according to claim 29 and neurons.

31. A vector comprising the nucleotide sequence encoding a dominant non-functional mutated form of astrocyte calcineurin under the control of a promoter.

32. Vector according to claim 30, wherein said promoter is a functional viral promoter in mammalian cells, preferably, a cytomegalovirus (CMV) promoter.

33. Vector according to claim 31, wherein said promoter is a specific astrocyte promoter, preferably, the promoter of the glial fibrillar acid protein

(GFAP).

34. A vector comprising calcineurin or an astrocyte calcineurin activator.

35. A method for obtaining a non-human animal model of a neurodegenerative disease comprising crossing a transgenic non-human animal according to any of claims 1 to 8, with an animal of the same species, either wild type (wt) or genetically manipulated with genes involved in neurodegenerative diseases, and separate useful animals as animal models of neurodegenerative diseases.

36. Non-human animal obtainable by the method of claim 35.