FR2810995A1 - COMPOSITIONS AND METHODS FOR THE TREATMENT OR DETECTION OF NEURODEGENERATIVE CONDITIONS - Google Patents

Abstract

The invention concerns the field of biology, genetics and medicine. In particular, it concerns novel methods for detecting, characterising and/or treating (or managing) degenerative diseases of the motor neurons, in particular amyotrophic lateral sclerosis. The invention also concerns methods for identifying or screening compounds active in said pathologies. The invention further concerns compounds, genes, cells, plasmids or compositions useful for implementing said methods. The invention finally concerns the part played by calcineurin in said pathologies and its use as therapeutic, diagnostic or experimental target.

Description

Compositions and methods for the treatment or detection of pathologies

  The present invention relates to the field of biology, genetics and medicine. It relates in particular to new methods for the detection, characterization and / or treatment (or management) of neurodegenerative pathologies, in particular of amyotrophic lateral sclerosis. The invention also relates to methods for

  the identification or screening of active compounds in these pathologies.

  The invention also relates to the compounds, genes, cells, plasmids or compositions useful for the implementation of the above methods. The invention describes in particular the role of calcineurin in these pathologies and its use

  as a therapeutic, diagnostic or experimental target.

  i5 Many neurodegenerative pathologies have been described as having a component or a stage linked to the phenomenon of excitotoxicity. This is the case of Alzheimer's disease, Parkinson's disease, multiple sclerosis

and Huntington's chorea.

  Amyotrophic lateral sclerosis (SAL or ALS for Amyotrophic Lateral Sclerosis) is a neurodegenerative disease, associated with different types of inclusions such as Lewis bodies and characterized by apoptosis of the spinal and cortical motoneurons, the fatal outcome of which is sometimes associated with frontal dementia. Sporadic forms, without any mutation described, coexist with familial forms (FALS) associated with mutations in the SOD1 gene coding for superoxide dismutase. The majority of cases are sporadic, familial forms (FALS) being very rare. It is likely that a long asymptomatic period precedes the onset of clinical symptoms which are varied and whose classification is complex. Future therapeutic developments will replace symptomatic treatments with strategies based on the molecular causes of the pathology. At the cellular level, these symptoms are associated with death of cortical motoneurons and spinal motoneurons. This neuronal death has been linked to different phenomena which constitute the basis of several neurodegenerative pathologies. This is the case of glutamate-related excitotoxicity, oxidative stress, a certain autoimmunity directed against neuronal markers (calcium channels in the case of ALS) as well as cytoskeletal abnormalities. If these phenomena are described, the cause or causes of these diseases, including ALS, are obscure. Even though FALS are linked to mutations in the SOD1 gene which codes for superoxide dismutase, the mechanisms which engage neurons towards cell death including at least

  îo one component is apoptosis are unknown.

  Identifying the molecular events involved in the various phenomena involved in cell death will allow new therapeutic strategies to be put in place. The study of these events is difficult to carry out from human biopsies. These biopsies obviously come from post-mortem samples whose quality is difficult to control and represent only pathological conditions representative of

late stages of the disease.

  Animal models provide access to biological samples which make it possible to analyze different stages of the development of a pathology and to compare these stages with healthy controls. In this regard, transgenic mice which express the human SOD1 gene carrying one of the mutations prevailing in FALS (mutation G93A) are available from Jackson Laboratory, subject to obtaining a license to use from NorthWestern University. This model reproduces in 120 days the fatal outcome of the

  disease with symptoms comparable to those of human disease.

  The appearance of ALS symptoms linked to the G93A mutation in SOD1 is not the consequence of a reduction in superoxide dismutase activity but of a gain in function which increases the capacity of the enzyme to generate radicals free. Despite this information, the molecular events that govern the different stages of ALS are poorly understood. The complexity of these molecular events reflects the evolution of the pathology: In the transgenic model studied, no neuronal deregulation or clinical manifestation was reported at 30 days. 60 days corresponds to a stage which slightly precedes the first symptoms, but which is already characterized in the brain by changes in cellular physiology such as an alteration in mitochondrial metabolism, stress and neuronal death associated with a phenomenon of excitotoxicity. At 90 days, 50% of cortical and spinal motor neurons are dead and an active process of neuronal apoptosis is initiated in parallel with astrocytic activation. The phenomenon of excitotoxicity is no longer observed at this stage. Neuronal death is associated with the activation of caspases which do not seem to be involved in the phases

early pathology.

  Identifying the different specific molecular events of the different phases of the pathology must make it possible to identify new therapeutic targets as well as new diagnostic markers. One of the most effective approaches to achieve this identification is to identify genes and proteins whose expression characterizes a pathophysiological state. The present invention now describes the identification of genetic events involved in the phenomena of excitotoxicity and neuronal death. The present invention thus provides new therapeutic and diagnostic approaches to the pathologies associated with these phenomena, as well as

  new targets for the identification of active compounds.

  More particularly, a qualitative differential analysis was carried out using RNA extracted from brain and spinal cord samples without prior isolation of the neurons in order to take into account a maximum

  alternative splicing events linked to the development of pathology.

  This analysis was carried out by qualitative differential screening according to the DATAS technique (described in application No. WO99 / 46403), which presents

unmatched benefits.

  The present patent application stems in particular from the construction by the applicant of a directory of splicing alterations in the brains of 60 day old ALS model animals. This repertoire, which contains more distinct sequences, involves key players in the phenomenon of excitotoxicity such as potassium channels and the NMDA receptor. Sequences derived from RNAs coding for proteins involved in the stress response, including heat shock proteins, are also part of this repertoire, highlighting the implication of this response in the early phases of ALS. An alteration in energy metabolism clearly appears to affect the cortical motor neurons of animals that develop the pathology. For example, intron 6 of the mitochondrial form of creatine kinase is isolated specifically from messenger RNAs expressed under pathological conditions in animals aged 60 days. This interruption in the coding sequence by this retention of intron results in a messenger RNA which codes for an inactive form of the enzyme. This observation is in agreement with biochemical observations which have shown a decrease in mitochondrial creatine kinase activity correlated with a decrease in the amount of

  this enzyme in the neurons of animals of the same transgenic model.

  The specificity of the sequences which constitute this repertoire is attested by the fact that the same qualitative differential analysis of the genetic expression carried out on animals aged 90 days leads to a different repertoire from which the various markers of excitotoxicity are absent in particular. Analysis of splice modifications confirms that molecular events are

  different depending on the stage of the pathology.

  Performing DATAS on 60-day-old control and transgenic animal RNA made it possible to isolate a cDNA fragment derived from the mRNA of the calcineurin catalytic subunit. This fragment corresponds to an exon fragment specifically present in the control animals and therefore specifically deleted in the transgenic animals for SOD1G93A at the 60 day stage. This fragment covers the nucleotides 1348 to 1579 referenced from the ATG. This region is essentially coding and contains an auto-inhibitory domain of the enzyme located at the Cterminal end of the enzyme. By RT PCR experiments, a new isoform of the calcineurin catalytic subunit has been demonstrated. This short isoform is distinguished from the long form by a deletion of nucleotides 1341 to 1743 and therefore amino acids 447 to 547, the stop codon of calcineurin corresponding to nucleotide 1641 (the complete sequence of calcineurin and its DNA, 0o murine or human origin, is available on database, in particular GeneBank). Mutagenesis and deletion experiments have shown that the calcineurin auto-inhibitory domain corresponds to amino acids 492 to 547 and that the absence of this domain results in constitutive activation of calcineurin. Calcineurin is involved in relaying the signaling pathways which depend on calcium and calmodulin. The interaction of calmodulin with calcineurin stimulates the activity of the latter. Deletion of the auto-inhibitory domain of calcineurin increases the basal level of activity of this enzyme but keeps it susceptible to activation by calmodulin. The present invention therefore describes an original and new molecular event (splicing), which results in constitutive activation of calcineurin and which is correlated with the phenomenon of excitotoxicity and / or neuronal death. The invention also shows, for the first time, that a constitutive activation of calcineurin is associated with the early stages of ALS. Calcineurin therefore constitutes a new and important therapeutic target in the development of therapeutics for these pathologies, usable in particular at the early stages of their development, and addressing the true molecular bases of the pathology and not the symptoms or components

associated immune systems.

  A first object of the invention lies more particularly in the use of a nucleic acid comprising all or part of a sequence derived from the messenger RNA of the catalytic subunit of calcineurin for the implementation of a method of diagnosing or detecting a situation of neuronal stress and more particularly the situation of excitotoxicity. The invention generally resides in the use of a nucleic acid complementary to all or part of the calcineurin gene or messenger, for the detection of pathological events of the excitotoxicity, stress or neuronal death type, etc. They are more particularly nucleic acids capable of demonstrating the deleted form of the calcineurin mRNA, in particular a splicing variant in which an auto-inhibitory domain is deleted, in particular residues 1341-1743 ( or the corresponding region of

gene or human DNA).

  Advantageously, the nucleic acid comprises all or part of the sequence coding the autoinhibiting domain of calcineurin (between nucleotides 1348 and 1579 from ATG, for the murine form, or homologous residues in the human sequence ), or of the sequence resulting from the junction between the non-deleted regions, or a complementary sequence

of these.

  The invention is applicable to the diagnosis or detection of various pathologies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's chorea or cerebral ischemia. It can be used for early detection, highlighting a predisposition, choosing and adapting a treatment, monitoring the evolution of the pathology, etc. It is particularly suitable for the detection at an early stage of

multiple sclerosis.

  Another object of the invention lies in the use of a compound capable of inhibiting or reducing the expression or the activity of calcineurin, for the preparation of a composition intended for the treatment of neurodegenerative diseases. In the context of the invention, the term “treatment” designates preventive, curative, palliative treatment, as well as the management of patients (reduction of suffering, improvement of lifespan, slowing down the progression of the disease ), etc. The treatment can also be carried out in combination with other agents or treatments, in particular addressing the late events of

  pathology, such as caspase inhibitors or other active compounds.

  In a particular embodiment, the compound is anti-nucleic acid

  sense, capable of inhibiting the transcription of the calcineurin gene or the translation of the corresponding messenger. The antisense nucleic acid may comprise all or part of the calcineurin gene sequence, a fragment thereof, the calcineurin messenger, or a sequence complementary thereto. The antisense can in particular comprise a region complementary to the form of splicing identified, and inhibit (or reduce) its

protein translation.

  According to another embodiment, the compound is a chemical compound, of natural or synthetic origin, in particular an organic or inorganic molecule, of vegetable, bacterial, viral, animal, eukaryotic, synthetic or semi-synthetic origin, capable of modulating expression or activity

calcineurin.

  As a preferred example, the compound is chosen from FK506 and cyclosporin A. Previous work carried out in vitro and in vitro using T lymphocytes has shown

  that superoxide dismutase (SOD) protects calcineurin from inactivation.

  However, a more recent study has shown that a mutated form of SOD1 which corresponds to a mutation found in familial forms of ALS does not have such an impact on calcineurin. A direct link between ALS and increase

  calcineurin activity is therefore excluded by these teachings.

  Several calcineurin inhibitors have been described in the prior art.

  s Some of these are commercially available drugs. This is the case of tacrolimus (or FK506) and cyclosporine A. These two compounds are immunosuppressants which bind to immunophilins, respectively the protein FKBP12 (FK506 binding protein 12) and cyclophilin A. Inhibition calcineurin, during the administration of these compounds, takes place by the formation of a complex between the compound, the immunophilin which it targets and the catalytic subunit of calcineurin. These immunophilins are peptidyl-prolyl-cis / transrotamases whose activity is also inhibited by their

chemical ligands.

  Some reports are available on the neuroprotective action of certain immunosuppressants in vitro. The most recent reports document that these neuroprotective effects, as well as the neurotrophic effects of these compounds are obtained with non-immunosuppressive immunophillins which have lost the capacity to inhibit calcineurin. Similarly, the results obtained in animals in ischemia confirm the results in vitro. Thus, no significant effect of the cyclosporine A or FK506 compounds has been described in an animal model of ALS or in patients suffering from this disease. To our knowledge, no trial has been carried out on the murine model and the absence of convincing results in humans is perfectly compatible with our observation that calcineurin is activated at the early stages of the development of pathology, during excitotoxicity. , and not at late stages. In the absence of an early diagnosis, the patients enrolled in the clinical study were patients with clinical manifestations and

  therefore probably engaged in the late stages of the disease.

  Today, there is therefore no evidence that inhibition of calcineurin by FK506 or cyclosporine A could slow the progression of ALS. On the other hand, it should be noted that no study has been undertaken in human pathology or using an animal model to take advantage of the

  ability of FK506 and cyclosporine A to inhibit the activity of calcineurin.

  Various trials, including those undertaken to treat neurodegenerative syndromes, tended to inhibit the immune component associated with

these pathologies.

  The present invention therefore provides, for the first time, calcineurin as a therapeutic target for the treatment of molecular events associated with exitotoxicity. According to particular embodiments, the invention can be used to inhibit or reduce neuronal excitotoxicity in the early phase of neurodegenerative diseases. It is applicable in particular to the treatment of Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's chorea or

cerebral ischemia.

  Other objects of the invention reside in: the use of the above compounds for the treatment of ALS, in particular for reducing neuronal excitotoxicity in the early phase of ALS, 20. the use of cyclosporine A for the preparation of a composition intended to inhibit the activity of calcineurin in patients suffering from ALS, or the use of FK506 for the preparation of a composition intended for

  inhibit calcineurin activity in patients with ALS.

  Other objects of the invention relate to methods of selection, identification, or characterization of compounds active on pathologies associated with excitotoxicity, or with neuronal stress, comprising bringing test compounds into contact with a cell expressing the variant d splicing of calcineurin lacking an auto-inhibitory domain, and the identification of

  compounds inhibiting the expression or activity of this protein.

  Another subject of the invention relates to a polypeptide derived from calcineurin, lacking the auto-inhibiting domain, in particular amino acids 492-547, preferably 447-547. This polypeptide can be of varied origin, in particular murine, human, synthetic or semi-synthetic, etc. Another subject of the invention relates to any nucleic acid encoding a polypeptide as defined above, the vectors containing it, cells

recombinant, and uses.

  Other aspects and advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting. Procedure: The qualitative differential analysis was carried out using poly adenylated RNA (poly A +) extracted from samples of animal brains corresponding to the different stages, without prior isolation of the neurons in order to take into account a maximum of alternative splicing events related to

pathology development.

  Poly A + RNAs are prepared according to techniques known to those skilled in the art. It may in particular be a treatment using chaotropic agents such as guanidium thiocyanate followed by extraction of the total RNAs using solvents (phenol, chloroform for example). Such methods are well known to those skilled in the art (see Maniatis et al., Chomczynsli et al., Anal. Biochem. 162 (1987) 156), and can be readily practiced using commercially available kits. From these total RNAs, poly A + RNAs are prepared according to conventional methods

  known to those skilled in the art and offered by commercial kits.

  These poly A + RNAs serve as a template for reverse transcription reactions using reverse transcriptase. Advantageously, reverse transcriptases devoid of RNase H activity are used which make it possible to obtain first strands of complementary DNA of sizes larger than those obtained II with conventional reverse transcriptases. Such reverse preparations

  transcriptases without RNase H activity are commercially available.

  For each point in the kinetics of pathology development (30 days, days and 90 days), poly A + RNAs as well as single-stranded cDNAs are prepared from transgenic animals (T) and control animals.

syngeneic (C).

  In accordance with the DATAS technique, for each point of the kinetics, hybridizations of mRNA (C) are carried out with cDNAs (T) and hybridizations

  reciprocal mRNA (T) with cDNA (C).

  o10 These mRNA / cDNA heteroduplexes are then purified according to the protocols of the

DATAS technique.

  RNA sequences not paired with complementary DNA are released from these heteroduplexes under the action of RNase H, this enzyme degrading the paired RNA sequences. These unpaired sequences represent the qualitative differences which exist between RNAs which are otherwise homologous to one another. These qualitative differences can be located anywhere on the RNA sequence, either in 5 ′, 3 ′ or within the sequence and in particular in the coding sequence. Depending on their location, these sequences can be not only splicing modifications but

  also consequences of translocations or deletions.

  The RNA sequences representing the qualitative differences are then cloned according to techniques known to those skilled in the art and in particular

  those described in the patent for the DATAS technique.

  These sequences are grouped within cDNA banks which constitute differential qualitative banks. One of these banks contains exons and introns specific to the healthy situation; the other banks contain

  splicing events characteristic of pathological conditions.

  The differential expression of the clones was verified by hybridization with probes obtained by reverse transcription from messenger RNA extracted from the different situations studied. The differential hybridization clones were retained for further analysis. The sequences identified by DATAS correspond to introns and / or exons expressed in a differential manner by splicing between the pathological situations and the healthy situation. These splicing events can be specific to a given stage of the

  development of pathology or characteristics of the healthy state.

  The comparison of these sequences with the databases makes it possible to classify the information obtained and to propose a reasoned selection of the

  sequences according to their diagnostic or therapeutic interest.

  This study made it possible to highlight a new isoform of the sub-

  catalytic unit of calcineurin, specific for pathological cells (or biological samples). This short isoform is distinguished from the long form by a deletion of nucleotides 1341 to 1743 and therefore amino acids 447 to 547, the stop codon of calcineurin corresponding to

nucleotide 1641.

  Administration of FK506 and Cyclosporin A to Mice

  Transgenic ALS models slow the progression of the pathology.

  The two compounds are administered according to three routes, intraperineal, intravenous and oral at different doses according to protocols known to those skilled in the art and derived from the protocols used to take advantage of their

  immunosuppressive properties, from animals aged 8 days.

  The results clearly indicate that the optimal result obtained with each of the compounds is a prolongation of the survival of these animals by 30 days.

  (i.e. a quarter compared to the control animals treated by the vehicle alone).

  Death occurs 150 days instead of 120 days. No modification of the kinetics is observed if the treatment is interrupted at 90 days, that is to say when the phenomenon of excitotoxicity is no longer present and when the constitutive activation of calcineurin is no longer revealed by detection of its isoform

  short deleted from its auto-inhibiting domain.

  The implication of calcineurin inhibition in the specificity of the therapeutic effect is underlined by the fact that (i) immunosuppressants which act by other mechanisms, in particular rapamycin, have no effect on the model studied (ii) a non-immunosuppressive derivative of cyclosporine which does not inhibit calcineurin but inhibits the romatase activity of cyclophilin

  A has no effect in the model studied.

  Other objects and applications of the invention reside in particular in: the use of all or part of a sequence derived from messenger RNA of the catalytic subunit of calcineurin for diagnostic or screening purposes or for characterization of neurodegenerative pathologies having a component or a stage linked to the phenomenon of excitotoxicity, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's chorea or cerebral ischemia, - the use of any nucleic acid fragment including antisense RNA is for the purpose of inhibiting the expression of calcineurin in patients suffering from such pathologies, the use of any chemical compound, in particular cyclosporin A or compound FK506, or of any pharmaceutical composition containing them, with the aim of inhibiting the activity of calcineurin in patients suffering from such pathologies, the use of all or part of a sequence derived from messenger RNA from the calcineurin catalytic subunit for characterization of tissue and ischemic status

Claims (13)

claims   1. Use of a nucleic acid comprising all or part of a sequence derived from messenger RNA of the catalytic subunit of calcineurin for the in vitro or ex vivo implementation of a diagnostic or detection method a situation of neuronal stress and more particularly the situation of excitotoxicity. 2. Use according to claim 1, characterized in that the nucleic acid 1o comprises all or part of the sequence coding the auto-inhibitory domain of calcineurin, or of the sequence resulting from the junction between the non-   deleted, or a sequence complementary thereto.   3. Use according to claim 1 or 2, for the diagnosis or detection of Alzheimer's disease, Parkinson's disease, multiple sclerosis,   Huntington's chorea or cerebral ischemia.   4. Use according to claim 1 or 2, for detection at an early stage multiple sclerosis.   5. Use of a compound which inhibits or reduces the expression or activity of calcineurin for the preparation of a composition intended for the treatment of neurodegenerative diseases.   6. Use according to claim 5, characterized in that the compound is antisense nucleic acid, capable of inhibiting the transcription of the gene for   calcineurin or the translation of the corresponding messenger.   7. Use according to claim 5, characterized in that the compound is a   chemical compound, of natural or synthetic origin.   8. Use according to claim 7, characterized in that the compound is chosen from FK506 and cyclosporin A.   9. Use according to one of claims 5 to 8, for inhibiting or reducing   Neuronal excitotoxicity in the early phase of neurodegenerative diseases.   s 10. Use according to one of claims 5 to 9, for the treatment of   Alzheimer's disease, Parkinson's disease, multiple sclerosis,   Huntington's chorea or cerebral ischemia.   11. Use according to one of claims 5 to 10, for the treatment of ALS,   o0 in particular to reduce neuronal excitotoxicity in the early phase of ALS.   12. Use of cyclosporin A for the preparation of a composition   intended to inhibit the activity of calcineurin in patients with ALS.   i5 13. Use of FK506 for the preparation of a composition intended for   inhibit calcineurin activity in patients with ALS.