EP1397485A2 - Populations de cellules destinees a la detection de cibles neuronales et de principes actifs potentiels - Google Patents

Populations de cellules destinees a la detection de cibles neuronales et de principes actifs potentiels

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Publication number
EP1397485A2
EP1397485A2 EP02754603A EP02754603A EP1397485A2 EP 1397485 A2 EP1397485 A2 EP 1397485A2 EP 02754603 A EP02754603 A EP 02754603A EP 02754603 A EP02754603 A EP 02754603A EP 1397485 A2 EP1397485 A2 EP 1397485A2
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EP
European Patent Office
Prior art keywords
cells
neuronal
receptors
population
substance
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02754603A
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German (de)
English (en)
Inventor
Thomas Rohrmeier
Rosemarie Daig
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Individual
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Individual
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Priority claimed from DE2001127008 external-priority patent/DE10127008C1/de
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Publication of EP1397485A2 publication Critical patent/EP1397485A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to novel cell populations which are suitable for finding neuronal targets for the investigation of neuronal diseases and potential pharmaceutical active substances, in particular against neuronal diseases, and to methods for finding such targets and active substances.
  • the brain is a precisely regulated network of more than 10 billion individual nerve cells.
  • Disorders of the complex regulatory mechanisms manifest themselves in neurodegenerative diseases such as Alzheimer's and Parkinson's disease or in neuropsychiatric diseases such as depression, schizophrenia and manic-depressive psychoses.
  • the causes of Alzheimer's and Parkinson's are still largely unknown.
  • Parkinson's dopaminergic neurons in the substantia nigra die and the formation of Lewybodies is observed.
  • the death of cholinergic neurons, the deposition of the amyloid-ß protein (Aß) and the hyperphosphorylation of the tau protein can be observed.
  • the drugs available so far cannot prevent the progression of these diseases and only temporarily alleviate the symptoms.
  • Neuropsychiatric disorders are believed to be due to neurotransmitter metabolic disorders such as dopamine and serotonin, glutamate, noradrenaline, and ⁇ -amino-butyric acid.
  • Known drugs are limited to the levels of the receptor blockade, the control of the tumor over rate and the resumption of the neurotransmitter into the cell.
  • Alternative concepts aim to protect nerve cells (nerve growth factors, neuroimmunophylline, antioxidants) or to replace them (cell transplantation).
  • New approaches include cell transplantation in Parkinson's and APP processing interventions, the use of anti-inflammatories and immunotherapy directed against ß-amyloid for Alzheimer's treatment.
  • New concepts in neuropsychiatric diseases are e.g. B. the administration of melatonin, CRF blockers, substance P antagonists, sigma receptor blockers, regulators of cannabinoid receptors and AMPAkinen. All previously known pharmaceutical agents against depression and schizophrenia influence the frequency of neurotransmitters in the brain. However, the success rates in treating these diseases with the known medications are low.
  • Patient material from post-mortem brain tissue is often used for target identification. Relevant genes are to be identified by comparing the gene expression in brains of healthy and diseased people.
  • the use of brain tissue as a basis for examination is also limited by degradation processes in post-mortem tissue and by adaptation processes that are caused in the brain by drug therapy.
  • the object of the present invention is to provide a cellular test system which is simple to manufacture and handle and at the same time is suitable for identifying and validating neural targets or active substances for the investigation or treatment of neuronal diseases.
  • the object is achieved with neuronal cell populations containing differentiated CNS neurons, which contain receptors from the group of the dopamine and serotonin receptors and the adrenergic receptors and which produce at least one neurotransmitter which does not bind to one of these receptors or does not produce them in undetectable amounts.
  • Such cell populations have proven to be a suitable cellular test system, with the identification or the investigation of target genes or target proteins with the controlled addition of the neurotransmitters that are not produced by the cell population or are produced in undetectable amounts.
  • the neuronal cell populations preferably contain further receptors from the group of GABA, acetylcholine, adenosine and glutamate receptors, in which case the cell populations do not produce at least one neurotransmitter that binds to one of these receptors or only produce them in undetectable amounts.
  • cell populations are suitable which have an increased surface density of the receptors, the neurotransmitters of which they bind do not produce them.
  • Preferred neuronal cell populations do not themselves produce neurotransmitters whose effects are to be investigated. However, undetectable amounts of neurotransmitters produced by the cell population can be tolerated.
  • the detection limits when checking the neurotransmitter content using ELISA are e.g. B. for dopamine at a concentration of 3.3 X 10 "10 M, for serotonin at 3.5 X 10 " 8 M and for norepinephrine at 3.5 X 10 "9 M. If these values are not exceeded, the reliability of the Gene induction experiments are not endangered if the respective neurotransmitter is added in a concentration that is above these limits by a factor of 10.
  • neurotransmitter concentrations produced by the cell populations are above the respective detection limits, it is recommended to inhibit the neurotransmitter production of the cell population by adding inhibitors Examples described here were cell culture samples containing 3 X 10 4 cells the production of dopamine, serotonin and noradrenaline tested by ELISA. After 24 hours, none of the neurotransmitters mentioned could be detected in the cell populations tested.
  • Neuronal cells are preferred for the receptors from the group dopamine D2-, serotonin (5HT-2A) -, -2-adrenergic, adenosine A2A-receptor are characteristic.
  • Cell populations which are obtained from neuronal primary cell cultures, from immortalized neuronal cell cultures or from neuronal cell lines are preferred, provided that they carry dopamine and serotonin receptors and adrenergic receptors.
  • Particularly preferred cell populations contain cells of the hNT type (NT-2 cells differentiated into neurons, for example described in Andrews, Dev. Biol., 103; 285-293, 1984; Andrews et al.: J. Lab.
  • Preferred cell populations furthermore consist of cells which can be generated and selected from one of the cell lines NT-2, SK-N-SH, SK-N-MC or SH-SY5Y. Neuronal cell populations which contain a high proportion of postmitotic differentiated CNS neurons are particularly preferred.
  • neurotransmitters are understood to be naturally occurring messenger substances which can trigger a receptor-bound signal cascade. In particular fall under the concept of
  • Neurotransmitters dopamine, serotonin, noradrenaline, glutamate, GABA, adenosine or acetylcholine. It should be noted that the invention neuronal cell populations may not only produce those neurotransmitters whose effects or whose signal cascade is to be investigated.
  • the neuronal cell populations according to the invention are added with the addition of substances having a differentiating action by culturing neuronal precursor cells, such as, for. B. by culturing NT-2 progenitor cells using retinoic acid.
  • Differentiating substances are e.g. B. phorbol esters, di-butyryl-cAMP, 1-isobutyl-3-methylxanthine, growth factors such as TGF-alpha, EGF, FGF2 or IGF-I or neurotrophins such as NT-3, BDNF or NGF.
  • Suitable differentiated CNS neurons are then selected on the basis of neuron-typical markers and on the basis of differentiation markers, preferably on the basis of the dopamine, serotonin and adrenergic receptors.
  • specific antibodies that recognize these receptors are used, which can be fluorescence-labeled or magnetically labeled.
  • Suitable CNS neurons are then selected using FACS or immunomagnetic
  • a typical neuron marker z. B. dopamine, serotonin, noradrenaline, glutamate, GABA, adenosine or acetylcholine receptors or a mixture of these receptors can be used.
  • the dopamine and serotonin receptors are suitable as differentiation markers.
  • the neural cell populations can then be cultivated in a serum-free or neurotransmitter-free medium. Cultures that do not produce neurotransmitters or only produce undetectable amounts of neurotransmitters are then checked by detecting any secreted neurotransmitters in the culture medium, e.g. B. by analytical methods such as HPLC, GC, MS or immunological methods such as ELISA or RIA.
  • tyrosine hydroxylase inhibitors such as ⁇ -methyl-DL-p-tyrosine to inhibit dopamine or norepinephrine biosynthesis or tryptophan hydroxylase inhibitors such as p-chlorophenylalanine and p-ethynylphenylalanine to inhibit serotonin biosynthesis, can be prevented efficiently.
  • the populations generated in this way with a high proportion of differentiated CNS neurons offer many advantages.
  • a cell culture system that is close to healthy nerve cells is available, which can be used to clarify the most important neuron-specific signaling pathways and their control with added chemical substances.
  • suitable target genes and target proteins can be identified that are involved in the development of neuronal diseases.
  • the effect of added potential active substances on the target genes and target proteins can be tested with the help of the cell populations.
  • the screening results obtained with the claimed neuronal cell populations are reproducible. This is also due to the fact that work is carried out in precisely defined cell populations, with false signals or interfering effects of undifferentiated neuronal cells or neuronal cells of another type being excluded.
  • the test conditions can still be precisely regulated.
  • the effect of an added neurotransmitter or another neuroactive substance can be enhanced due to the increased receptor density on the cell surfaces.
  • the claimed cell cultures are easy to cultivate.
  • the neuronal cell populations enable the investigation of pathological processes starting with healthy neurons. It can be advantageous to grow the neuronal cell populations in a coculture with other cells of the nervous system in order to create a cellular environment that is as natural as possible.
  • Cells of the nervous system that are suitable for such cocultivation are neurons or glial cells, such as B. astrocytes, oligodendrocytes, microglial cells, or a combination of these cell types.
  • the present invention further relates to methods for finding target genes or target proteins.
  • a population according to the invention of differentiated neuronal cells or differentiated hNT, SK-N-SH, SK-N-MC or SH-SY5Y cell populations is cultivated in a serum-free or neurotransmitter-free medium.
  • the serum-free cultivation of neuronal cells is e.g. B. described in US 6 180 404 or in US 6 040 180.
  • the cell populations according to the invention can be cultivated in simple media made from Dulbecco's MEM / Nutrient Mixture F-12 (Ham) (DMEM / F12), insulin transferrin and selenium.
  • Target genes can then be identified by determining the change in the transcription rate of the total cell transcript, that is to say the entire mRNA isolated from the cell population, or one or more defined genes.
  • the mRNA of the neuronal cell population is isolated and, if desired, converted into cDNA.
  • the mRNAs or cDNAs can then be separated and determined quantitatively, it being advantageous to also determine the mRNA or cDNA of a reference population that was not exposed to a potentially neuroactive substance and to use it for comparison.
  • Potential target genes can also be determined by forming a subtractive cDNA library, the transcription rate of the neuronal population being compared with that of the reference population.
  • Target proteins can be identified by determining the change in the expression rate of the entire proteome or one or more defined proteins.
  • the determination can also be determined here in relation to the protein content in a reference population.
  • the determination of the protein content by means of 2D gel electrophoresis is particularly suitable, the proteins from the neuronal population being able to be separated electrophoretically together with differently labeled proteins, isolated from the reference population.
  • this method can also be used to investigate the effect of potentially neuroactive substances on a target gene or on a target protein.
  • the change in the transcription of the target gene or the change in protein expression is determined depending on the potentially neuroactive substance or mixture of substances.
  • neuroactive substances in particular neurotransmitters, such as. B. dopamine, serotonin, noradrenaline, glutamate, GABA, adenosine or acetylcholine, their agonists or antagonists understood.
  • the term also includes synthetic derivatives or analogues of these substances that are to be examined for their neuroactive effects.
  • Neuroactive substances can also oxidizing or reducing substances, such as. B. H 2 O 2 , vitamin C, vitamin E or estrogens. Mixtures of neuroactive substances can also be examined.
  • the neuroactive substances are usually added in a concentration of 1 X 10 "10 M to 1 X 10 " 3 M. Typical induction times are between one minute and 4 weeks, preferably at a temperature between 35 ° C and 38 ° C, in an incubator with a carbon dioxide content between 2.5% and 15%.
  • Typical induction times are between one minute and 4 weeks, preferably at a temperature between 35 ° C and 38 ° C, in an incubator with a carbon dioxide content between 2.5% and 15%.
  • the present invention furthermore relates to mixtures comprising neuronal cell populations according to the invention and the neuroactive substances or substance mixtures to be tested or cells which secrete such substances or substance mixtures.
  • Mixtures are preferred which have at least one neurotransmitter, such as. B. Contain dopamine, serotonin, noradrenaline, GABA, acetylcholine, adenosine, glutamate or a mixture of these neurotransmitters.
  • Mixtures which also contain a neurotransmitter agonist or antagonist are also preferred.
  • the mixtures are advantageously prepared in a serum-free or neurotransmitter-free medium.
  • heterologous genes which can be transcribed with vectors on the cell population according to the invention can also be investigated.
  • Another object of the present invention is a cellular
  • Screening system e.g. B. in the form of a kit, in which the claimed neural cell populations are contained.
  • Such cellular screening systems preferably contain a set of neurotransmitters which bind specifically to the selected receptors and can thus induce a neuron-typical signal in a controlled manner.
  • serum-free or neurotransmitter-free media can be added to the cellular screening system for the cultivation of the claimed neuronal cell populations.
  • Example 1 Production of the differentiated, neuronal cell population
  • NT precursor cells (Ntera2 / clone D1) (Stratagene) were induced for five weeks with 10 ⁇ M all-trans retinoic acid in DMEM / F12, supplemented with 10% FCS and 1% penicillin streptomycin (Andrews, PW, Dev. Biol ., 103, 285-293, 1984; Andrews et al, J. Lab. Invest, 50; 147-162, 1984).
  • the differentiation was checked by the presence of ⁇ -intemexin (Neurofilament 66), a type IV intermediate filament specific for mature CNS neurons, by means of Western blot.
  • 10 ⁇ g protein from differentiated NT neurons were separated on a 10% SDS gel, transferred to a PVDF membrane and incubated with an anti- ⁇ -internexin antibody.
  • the cells were treated with verse (1: 5000), trypsinized (0.05% trypsin / 0.53 mM EDTA) and mechanically separated from each other (pipetting up and down).
  • the cells were spurted 1: 6 and cultured in DMEM / F12, with 10% FCS and 1% penicillin-streptomycin for 2 days.
  • Morphological assessment 5% of the cells are round, glowing cells and correspond to the neuron phenotype. By trypsinizing for one minute (0.05% trypsin / 0.53 mM EDTA), mainly differentiated cells were detached.
  • an immunomagnetic selection method (Dynabeads) was used.
  • the cells were selected with antibodies against the differentiation markers dopamine D2 receptor and against serotonin receptor 5-HT2A and with the neuron-typical antibodies against the ⁇ 2-adrenergic receptors and against the adenosine receptor A2A.
  • the Eppendorf Cup with the suspension of cells and bead-coupled antibodies was positioned during the immunomagnetic selection process 1 cm from the Dynal magnet with a field strength of 1, 220 mTesla and waited for one minute until the Cells with the highest receptor density, ie cells with the most bead-coupled antibodies, have placed on the cup wall.
  • the suspension in the cup with the cells with a low receptor density was aspirated and discarded.
  • the cells with high receptor density are the differentiated neurons that are used for the gene induction experiments.
  • the vitality of the cells is> 95%. This cell population was sown on poly-D-lysine and Matrigel (1:30) coated cell culture dishes (1 - 2 x 10 4 per cm 2 ).
  • This population of differentiated neurons was first exposed to DMEM / F12 + 10% FCS + mitosis inhibitors (10 ⁇ M 5-fluoro-2-deoxyuridine, 10 ⁇ M uridine, 1 ⁇ M cytosine- ⁇ -D-arabinofuranoside) for 10 days and then for 1 day each cultivated in DMEM / F12 with 2% FCS or 0.5% FCS.
  • the lines were checked morphologically on day 13 after sowing: over 99% of the cells formed the neuron-typical offshoots.
  • the cell culture medium used did not contain any neurotransmitters and that the neuronal cell population itself did not produce any neurotransmitters.
  • the neuronal cell population was kept in serum-free medium from day 13 after sowing, to exclude effects from neurotransmitters in the serum.
  • the serum-free medium consists of DMEM / F12, 2.5 ⁇ g / ml insulin, 2.5 ⁇ g / ml transferrin and 2.5 ng / ml selenium. Since neurotransmitters max. 1000 Da are large, alternatively the serum was dialyzed with a dialysis tube with an exclusion volume of 1000 Da and 0.5% added to the medium.
  • the cell population is adapted in the above-mentioned serum-free or neurotransmitter-free medium for 3 days and kept in this medium during the gene expression experiment. In order to ensure constant concentrations of the neuroactive substances, the medium was changed daily and the neuroactive substances were added freshly.
  • control cells were treated identically, but without adding the
  • RNA isolation is carried out according to P. Chomzynski and N. Sacchi (Anal. Biochemistry 162, 156-159, 1987). The isolated RNA is dissolved in H 2 0 and its amount determined spectrophotometrically.
  • Total RNA was used to determine differences in the abundance of specific m-RNAs that were regulated by the neuroactive substances. For this, the total RNA (1 mg / ml) was first treated with DNase I (50 U / ml) at 37 ° C. for 30 min, then 10 ⁇ DNase termination mix was added, the same volume of phenol: chloroform: isoamyl alcohol (25:24: 1) added, mixed, centrifuged for 10 min to separate the phases (14000 rpm microcentrifuge) and the supernatant extracted again with chloroform. By adding 1/10 volume The RNA was precipitated on ice for 2 min with 2 M Na acetate (pH 4.5) and 2.5 volumes of ethanol. The DNase I-treated RNA was centrifuged at 14,000 rpm for 15 min, washed with 70% ethanol, dried and dissolved in H 2 O.
  • the first strand of cDNA is synthesized with oligo-dT ⁇ 2- ⁇ 8 , MMLV reverse transcriptase (20 U / ⁇ l) (1 hour, 37 ° C.).
  • This cDNA first strand was used according to Gubler & Hofmann (Gene, 25, 263, 1983) for the cDNA second strand synthesis.
  • a cDNA library was then synthesized, which was subjected to differential hybridization.
  • the MAP kinase-activated protein kinase-2 (Accession number U 12779) was found as a regulated gene in a macroarray experiment.
  • the neuronal cell population was incubated in serum-free medium with 50 ⁇ M dopamine (dissolved in PBS w / o Ca 2+ Mg 2+ ).
  • Control cells were treated with PBS in serum-free medium.
  • the total RNAs were prepared and in each case> 15 ⁇ g of them were used in the production of the gene-specific hybridization probe radioactively labeled with ⁇ - 32 P-dATP.
  • a dopamine-induced reduction in the expression of the MAP kinase-activated protein kinase-2 by 30% was found.

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Abstract

L'invention concerne des populations de cellules neuronales contenant les récepteurs du groupe des récepteurs de dopamine, des récepteurs de sérotonine et des récepteurs adrénergiques et ne produisant pas au moins un neurotransmetteur se liant à ces récepteurs. Ces populations de cellules permettent de détecter des cibles neuronales permettant de déceler des maladies neuronales, et des principes pharmaceutiques actifs potentiels agissant notamment contre des maladies neuronales. L'invention concerne également un procédé de détection de telles cibles et de tels principes actifs.
EP02754603A 2001-06-05 2002-05-31 Populations de cellules destinees a la detection de cibles neuronales et de principes actifs potentiels Withdrawn EP1397485A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10127008 2001-06-05
DE2001127008 DE10127008C1 (de) 2001-06-05 2001-06-05 Zellpopulationen zur Auffindung neuronaler Targets und potentieller Wirkstoffe
DE10216045 2002-04-11
DE10216045 2002-04-11
PCT/EP2002/005968 WO2002099087A2 (fr) 2001-06-05 2002-05-31 Populations de cellules destinees a la detection de cibles neuronales et de principes actifs potentiels

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EP1397485A2 true EP1397485A2 (fr) 2004-03-17

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EP (1) EP1397485A2 (fr)
WO (1) WO2002099087A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5654189A (en) * 1991-10-21 1997-08-05 Trustees Of The University Of Pennsylvania Preparation of pure cultures of post-mitotic human neurons

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2662697A (en) * 1996-04-09 1997-10-29 Board Of Trustees Of Southern Illinois University, The A cultural medium for maintaining neural cells in ambient atmosphere
US5753506A (en) * 1996-05-23 1998-05-19 Cns Stem Cell Technology, Inc. Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5654189A (en) * 1991-10-21 1997-08-05 Trustees Of The University Of Pennsylvania Preparation of pure cultures of post-mitotic human neurons

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BORLONGAN C.V. ET AL: "Transplantation of Cryopreserved Human Embryonal Carcinoma-Derived Neurons (NT2N Cells) Promotes Functional Recovery in Ischemic Rats", EXPERIMENTAL NEUROLOGY, vol. 149, no. 2, February 1998 (1998-02-01), pages 310 - 321, XP000961826, DOI: doi:10.1006/exnr.1997.6730 *
GUILLEMAIN I. ET AL: "Human NT2 Neurons Express a Large Variety of Neurotransmission Phenotypes In Vitro", THE JOURNAL OF COMPARATIVE NEUROLOGY, vol. 422, no. 3, 3 July 2000 (2000-07-03), pages 380 - 395, XP009043336 *
See also references of WO02099087A3 *

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WO2002099087A3 (fr) 2003-04-10

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