DE10313098A1 - Test system for finding active substances against prion-induced diseases and active substances for the prevention and treatment of this disease - Google Patents
Test system for finding active substances against prion-induced diseases and active substances for the prevention and treatment of this disease Download PDFInfo
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- DE10313098A1 DE10313098A1 DE2003113098 DE10313098A DE10313098A1 DE 10313098 A1 DE10313098 A1 DE 10313098A1 DE 2003113098 DE2003113098 DE 2003113098 DE 10313098 A DE10313098 A DE 10313098A DE 10313098 A1 DE10313098 A1 DE 10313098A1
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- nerve cell
- prion protein
- prpc
- active substances
- test system
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Abstract
Die Erfindung betrifft ein Testsystem zur Auffindung von Wirksubstanzen, welche die Hemmung des Axonwachstums durch infektiöse Prionen aufheben, mit den folgenden Komponenten: a) einer in Kultur gehaltenen Nervenzelle, wobei diese Nervenzelle in der Zellkultur Axone ausbildet, b) eine Zubereitung, enthaltend abnormales Prion-Protein, und c) ein Nachweissystem für die Ausbildung von Axone, ein Verfahren zur Auffindung von Wirksubstanzen, welche die Hemmung des Axonwachstums durch infektiöse Prionen aufheben, mittels eines solchen Testsystems, sowie damit ermittelbare Wirksubstanzen.The invention relates to a test system for the detection of active substances which cancel the inhibition of axon growth by infectious prions, with the following components: a) a nerve cell kept in culture, this nerve cell forming axons in the cell culture, b) a preparation containing abnormal prion -Protein, and c) a detection system for the formation of axons, a method for the detection of active substances which remove the inhibition of axon growth by infectious prions, by means of such a test system, and active substances which can thus be determined.
Description
Gebiet der Erfindung.Field of the Invention.
Die Erfindung betrifft ein Testsystem sowie ein Verfahren zur Identifizierung von Wirksubstanzen, welche zur Prophylaxe und/oder Behandlung von Prionerkrankungen geeignet sind. Die Erfindung betrifft weiterhin solche Wirksubstanzen sowie pharmazeutische Zusammensetzungen und Formulierungen enthaltend solche Wirksubatnzen.The The invention relates to a test system and a method for identification of active substances which are used for the prophylaxis and / or treatment of Prion diseases are suitable. The invention further relates to such active substances and pharmaceutical compositions and Formulations containing such active substances.
Stand der Technik und Hintergrund.State of the art and Background.
Prionerkrankungen oder transmissible Spongiforme Encephalopathien (TSE) wie beispielsweise Scrapie (SC), die Bovine spongiforme Encephalopathie (BSE) und die Creutzfeldt-Jakob Erkrankung (CJD) stellen Erkrankungen des zentralen Nervensystems dar, die mit Vakuolierung, Gliose und einer Anreicherung einer krankheitsspezifischen, Protease resistenten, β-gefalteten Isoform (PrPSc; PrPCJD) des normalen Prion-Proteins (PrPc) einhergehen.prion diseases or transmissible spongiform encephalopathies (TSE) such as scrapie (SC), Bovine Spongiform Encephalopathy (BSE) and Creutzfeldt-Jakob Disease (CJD) represent diseases of the central nervous system represents that with vacuolation, gliosis and an enrichment of a disease-specific, Protease resistant, β-folded Isoform (PrPSc; PrPCJD) of normal prion protein (PrPc) is associated.
Das PrPc ist ein Glykoprotein, welches auf der Membran fast aller Zellen, besonders der Nervenzellen anzutreffen ist und an der Membran über einen Glycosylphosphatidylinositol- Anker verbunden ist. In den Nervenzellen kommt das PrPc in höherer Konzentration vor und ist hier besonders in den präsynaptischen Membranen anzutreffen (Ferrer et al Neuroscience 97: 71-726, 2000).The PrPc is a glycoprotein that is on the membrane of almost all cells, especially the nerve cells and on the membrane over one Glycosylphosphatidylinositol anchor is connected. In the nerve cells the PrPc comes in higher concentration before and can be found particularly in the presynaptic membranes (Ferrer et al Neuroscience 97: 71-726, 2000).
Die Funktion des PrPc ist noch nicht eindeutig geklärt. Es bestehen Anhaltspunkte, dass PrPc eine Rolle im Kupfermetabolismus spielt, und dass Kupfer-Ionen die Endozytose von PrPc stimulieren. Andere Studien weisen auf eine Rolle von PrPc beim circadianen Rhythmus hin (Tobler et al., 1996), und bei der Langzeitpotenzierung von Synapsen (Collinge et al., 1994). Des Weiteren scheint PrPc ein hoch affiner Ligand für Laminin zu sein und hierdurch die Adhesion und Präsens von Nervenzellen und das Streckenwachstum von Neuriten zu unterstützen (Martins et al Braz.J Med Biol Res 34: 585-595; 2001. Zusätzlich führt die Bindung von PrPc an Caveolin-1 zur Aktivierung von zellulären signalübertragenden Kinasen, wie beispielsweise von Fyn (Mouillet-Richard et al., 2000); Kellermann et al CR Acad Sci 325: 9-15, 2002). Da PrPc, beispielsweise nach Kreuzvernetzung über Antikörper, auch den cAMP/PKA- abhängigen Signalweg aktiviert, wird dem PrPc auch eine Funktion als neurotropher Rezeptor zugeschrieben, dessen Aktivierung eine neuroprotektive Wirkung aufweisen soll (Chiarini et al EMBO 21: 3317-3327; 2002).The The function of the PrPc has not yet been clearly clarified. There are indications that PrPc plays a role in copper metabolism, and that copper ions stimulate endocytosis of PrPc. Other studies point to one Role of PrPc in the circadian rhythm (Tobler et al., 1996), and in the long-term potentiation of synapses (Collinge et al., 1994). PrPc also appears to be a highly affine ligand for laminin to be and thereby the adhesion and presence of nerve cells and the route growth of neurites (Martins et al Braz. J Med Biol Res 34: 585-595; 2001. In addition, the binding of PrPc leads Caveolin-1 for the activation of cellular signal-transmitting kinases, such as by Fyn (Mouillet-Richard et al., 2000); Kellermann et al CR Acad Sci 325: 9-15, 2002). Since PrPc, for example after cross-linking via antibodies, too the cAMP / PKA-dependent Activated signal path, the PrPc is also a function as a neurotrophic Attributed receptor, its activation is a neuroprotective Should have an effect (Chiarini et al EMBO 21: 3317-3327; 2002).
Es wird angenommen, dass eine Infektion mit abnormalem, Protease-resistenten PrPSC die Ursache der TSE ist. Befunde, dass eine chemische oder immunologische Sympathektomie nach i.p. Injektion von PrPsc den Ausbruch eine TSE verzögern kann, legen nahe, dass die Infektion des ZNS durch abnormale Prionen durch eine Neuroinvasion erfolgen kann (Glatzel et al Neuron 31: 25-34, 2001)It is believed to be an infection with abnormal, protease-resistant PrPSC is the cause of the TSE. Finds that a chemical or immunological sympathectomy after i.p. Injection of PrPsc Outbreak of a TSE can delay suggest that the CNS infection is due to abnormal prions a neuroinvasion can occur (Glatzel et al Neuron 31: 25-34, 2001)
Nach Infektion reichert sich PrPSC in Nervenzell-Ausläufern und in den Synapsen an (Ferrer et al Neuroscience 97: 715-726, 2000). Das abnorme PrP (PrPSC; PrPCJD) interagiert mit dem PrPc und führt zu einer strukturellen Umformung des PrPc.To Infection accumulates in PrPSC in nerve cell extensions and in the synapses (Ferrer et al Neuroscience 97: 715-726, 2000). The abnormal PrP (PrPSC; PrPCJD) interacts with the PrPc and leads to one structural reshaping of the PrPc.
Im Zuge dieser Anreicherung von abnormalem PrP sind Degenerationen der Axon-Enden und von Dendriten beschrieben worden (Jeffrey et al Neuropathol Appl Neurobiol 26: 41-54, 2000; Jeffrey et al Neuropathol Appl Neurobiol 23: 93-101, 1997).in the In the course of this accumulation of abnormal PrP are degenerations of the axon ends and dendrites (Jeffrey et al Neuropathol Appl Neurobiol 26: 41-54, 2000; Jeffrey et al Neuropathol Appl Neurobiol 23: 93-101, 1997).
Wie jedoch das abnormale PrP zur TSE führt, ist weitgehend unbekannt.How however, the abnormal PrP leading to TSE is largely unknown.
Derzeit gibt es keine Möglichkeit einer effektiven Therapie der TSE (Collins et al (2002) Ann. Neurol 52: 503-506). Die bislang versuchten Strategien zur Therapie der TSE beinhalteten im wesentlichen die Inhibition der durch PrPSC oder PrPCJD verursachten Konversion des PrPc in ein abnormales PrPSC durch spezifische RNA Aptamere (Proske et al; (2002) Chembiochem 3, 717-725); Quinacrine (Caughey et al (2002) Biochem Soc Trans 30: 565-569; Collins et al (2002) Ann. Neurol 52: 503-506); durch Fragmente der Prion-Protein Isoformen und durch Peptidomimetika (Horiuchi et al (2001) J Biol Chem 276: 15489-15497; Chabry et al (1998), J Biol Chem 273: 13203-13207); durch Kongorot und Analoge hiervon (Rudyk et al (2000) J Gen Virol 81: 1155-1164), Porphyrine und Phtalocyanine (Caughey et al (1998) PNAS USA 95: 12117-12122) Tetrazyclinderivate (Forloni et al., 2002) oder durch Triaminopyridine (Perovic et al (1995), Neurodegeneration 4: 369-374.Currently is there no possibility an effective therapy for TSE (Collins et al (2002) Ann. Neurol 52: 503-506). The strategies for the therapy of TSEs essentially involved the inhibition of PrPSC or PrPCJD caused conversion of the PrPc into an abnormal PrPSC by specific RNA aptamers (Proske et al; (2002) Chembiochem 3, 717-725); Quinacrine (Caughey et al (2002) Biochem Soc Trans 30: 565-569; Collins et al (2002) Ann. Neurol 52: 503-506); by Fragments of prion protein isoforms and by peptidomimetics (Horiuchi et al (2001) J Biol Chem 276: 15489-15497; Chabry et al (1998), J Biol Chem 273: 13203-13207); by Congo red and analogues thereof (Rudyk et al (2000) J Gen Virol 81: 1155-1164), porphyrins and phthalocyanines (Caughey et al (1998) PNAS USA 95: 12117-12122) tetracycline derivatives (Forloni et al., 2002) or by triaminopyridines (Perovic et al (1995), Neurodegeneration 4: 369-374.
In einem besonderen Forschungsansatz wurde die Wechselwirkung zwischen PrPc und abnormalem PrPSC durch Antikörper gegen PrP in vitro (Peretz et al (2001) Nature 16/412: 739-743, (Enari et al., 2001) oder in vivo Koller et al J Neuroimmunol (2002) 132: 113-116; White et al (2003), Nature 422: 80-83 oder durch Immunisierung gegen PrP in einem transgenen Mausmodell (Heppner et al (2001) Science; 5/294 178-182, Sigurdson et al Neurosci Lett (2003), 336: 185-187) inhibiert und hierdurch eine Verzögerung im Auftreten der TSE bei der Maus bewirkt.In the interaction between PrPc and abnormal PrPSC by antibodies against PrP in vitro (Peretz et al (2001) Nature 16/412: 739-743, (Enari et al., 2001) or in vivo Koller et al J Neuroimmunol (2002) 132: 113-116; White et al (2003), Nature 422: 80-83 or by immunization against PrP in a transgenic mouse model (Heppner et al (2001) Science; 5/294 178-182, Sigurdson et al Neurosci Lett (2003), 336: 185-187) and thereby a delay in the occurrence of the TSE in the mouse.
Allein jedoch der Befund, dass der Neurotrophin p75 Rezeptor an der Neurotoxizität von abnormalem Prion-Protein entscheidend beteiligt zu sein scheint (Della-Bianca et al (2002) J Biol Chem 19/276: 38929-389233) und dass Cyclooxygenase Inhibitoren die Neurotoxizität von abnormalen Prion-proteinen reduzieren können (Bate et al (2002) Neuroreport 28/13 1933-1938) verdeutlicht, dass die Pathophysiologie der Neurotoxizität des abnormalen Prion-Proteins nicht alleine auf seine Wechselwirkung mit dem PrPc reduziert werden kann.However, the finding alone that the neurotrophin p75 receptor appears to be crucially involved in the neurotoxicity of abnormal prion protein (Della-Bianca et al (2002) J Biol Chem 19/276: 38929-389233) and that cyclooxygenase Inhibitors that can reduce the neurotoxicity of abnormal prion proteins (Bate et al (2002) Neuroreport 28/13 1933-1938) clarify that the pathophysiology of the neurotoxicity of the abnormal prion protein cannot be reduced to its interaction with the PrPc alone.
In Anbetracht dieser lückenhaften Kenntnis über die pathophysiologische Wirkung des abnormalen Prion-Proteins in der Entstehung einer TSE besteht ein erheblicher Bedarf nach neuen Erkenntnissen in der Entstehung der TSE, auf Basis derer dann Testsysteme aufgebaut werden können für die Suche nach kausal wirkenden Substanzen für die Prophylaxe und frühe Behandlung einer TSE.In Given this sketchy Knowledge of the pathophysiological effect of the abnormal prion protein in the emergence of a TSE there is a considerable need for new ones Insights into the emergence of TSE, on the basis of which test systems are then based can be built for the Search for causal substances for prophylaxis and early treatment a TSE.
Technisches Problem der Erfindung.Technical problem of the Invention.
Daher liegt der Erfindung das technische Problem zu Grunde, Mittel anzugeben, mit welchen Wirksubstanzen zur Prophylaxe und/oder Behandlung einer TSE identifiziert werden können, sowie solche Wirksubstanzen.Therefore the invention is based on the technical problem of specifying means with which active substances for the prophylaxis and / or treatment of a TSEs can be identified as well as such active substances.
Der Erfindung zu Grunde liegende Erkenntnisse.The Findings on which the invention is based.
Es wurde überraschend gefunden, i) dass das Wachstum von Axonen in Kulturen von Motoneuronzellen von Mäusen (Prnp –/–), in denen das Gen für das PrPc genetisch deletiert worden war, deutlich geringer ist als in Mäusen vom Wildtyp. Diese Verminderung des Axonwachstums der Motoneuronen von Prnp (–/–) Mäusen tritt unabhängig davon auf, welche neurotrophen Faktoren (BDNF; CNTF, GDNF) der Motoneuronkultur hinzugegeben werden, ii) dass im Gegensatz zum verringerten Wachstum der Axone das Wachstum der Motoneurone und ihrer Dendriten nicht unterschiedlich ist zwischen Mäusen vom Wildtyp (Prnp +/+) und Mäusen mit Deletion des Gens für PrPc (Prnp –/–), iii) dass in embryonalen Motoneuronen von (Prnp +/+) Mäusen das endogene PrPc mit Tau, einem in Axonen lokalisiertem Protein, kolokalisiert, und iv) dass die Zugabe von infektiösen Hirnhomogenaten (PrpSC enthaltend) zu Motoneuronkulturen von (Prnp +/+) Mäusen zu einer drastischen Hemmung des Axonwachstums führt wie auch zu einem dosisabhängigen Absterben der Motoneuronen.It became surprising found i) that the growth of axons in cultures of motor neuron cells of mice (Prnp - / -) in which the gene for the PrPc had been genetically deleted is significantly less than in mice of the wild type. This decrease in axon growth of the motor neurons from Prnp (- / -) mice occurs independently on which neurotrophic factors (BDNF; CNTF, GDNF) of the motor neuron culture be added, ii) in contrast to the reduced growth the axons do not support the growth of the motor neurons and their dendrites is different between mice from Wild type (Prnp + / +) and mice with deletion of the gene for PrPc (Prnp - / -), iii) that in embryonic motor neurons of (Prnp + / +) mice endogenous PrPc with tau, a protein localized in axons, colocalized, and iv) that the addition of infectious brain homogenates (PrpSC containing) to motor neuron cultures of (Prnp + / +) mice drastic inhibition of axon growth leads to dose-dependent death of the motor neurons.
Es kann somit aus diesen Ergebnissen geschlussfolgert werden, i) dass das PrPc ein entscheidender Faktor für das Wachstum von Axonen ist, ii) dass der Kontakt von Motoneuronen mit Prionen das Axonwachstum von Motoneuronen drastisch hemmt und zum Absterben der Motoneuronen führt und iii) dass eine Hemmung des Kontaktes zwischen Motoneuronen einschließlich Ihrer Ausläufer mit abnormalem Prion-Protein zu einer Verhinderung der Hemmung des Axonwachstums und des Absterbens von Motoneuronen führt.It can be concluded from these results: i) that that PrPc is a crucial factor for the growth of axons, ii) that the contact of motor neurons with prions inhibits axon growth of motor neurons drastically inhibits and the motor neurons die leads and iii) an inhibition of contact between motor neurons, including yours offshoot with abnormal prion protein to prevent the inhibition of axon growth and death of motor neurons.
Grundzüge der Erfindung sowie bevorzugte Ausführungsformen.Basics of the invention as well as preferred embodiments.
Gegenstand der Erfindung ist somit ein Testsystem zur Auffindung von Wirksubstanzen, welche die Hemmung des Axonwachstums durch Prionen aufheben, bestehend aus
- – einer in Kultur gehaltenen Nervenzelle, im besonderen einem Motoneuron, wobei diese Nervenzelle fakultativ PrPc exprimiert und Axone in der Zellkultur ausbildet und
- – infektiöses Hirnhomogenat, welches abnormales Prion-Protein, beispielsweise PrPSC oder PrPCJDenthält
- – und einem Nachweissystem für die Ausbildung von Axone, wobei dieses Nachweissystem die Länge des gebildeten Axons pro Nervenzelle direkt oder beispielsweise über den Nachweis von Tau erfasst.
- A nerve cell kept in culture, in particular a motor neuron, which nerve cell optionally expresses PrPc and forms axons in the cell culture and
- Infectious brain homogenate containing abnormal prion protein, e.g. PrPSC or PrPCJD
- - And a detection system for the formation of axons, this detection system detects the length of the axon formed per nerve cell directly or, for example, by detecting dew.
Gegenstand der Erfindung ist des Weiteren ein Verfahren zur Auffindung von Wirksubstanzen, welche die Hemmung des Axonwachstums durch Prionen aufheben, bei welchem
- – eine Prüfsubstanz in Kontakt gebracht wird mit einer Nervenzelle, im besonderen einer Motoneuronzelle, welche fakultativ PrPc exprimiert
- – und geprüft wird, ob nach Zugabe von Prionen diese Prüfsubstanz in der Lage ist, die Hemmung des Axonwachstums durch Prionen und das Absterben der Nervenzelle zu verhindern,
- – wobei als Parameter die Länge des in der Zellkultur ausgebildeten Axons einer Nervenzelle direkt oder über den Nachweis von Tau erfasst wird.
- - A test substance is brought into contact with a nerve cell, in particular a motor neuron cell, which optionally expresses PrPc
- - and it is checked whether, after the addition of prions, this test substance is able to prevent the inhibition of axon growth by prions and the death of the nerve cell,
- - The length of the axon of a nerve cell formed in the cell culture is recorded as a parameter, either directly or via the detection of tau.
Gegenstand der Erfindung sind des Weiteren Wirksubstanzen, welche in dem erfindungsgemäßen Testsystem oder in dem erfindungsgemäßen Verfahren die Wirkung des abnormalen Prion-Proteins auf das Axonwachstum und auf das Überleben der Nervenzelle verhindern können und die Verwendung dieser Wirksubstanzen zur Vorbeuge oder Behandlung einer TSE.object The invention furthermore contains active substances which are in the test system according to the invention or in the method according to the invention the effect of the abnormal prion protein on axon growth and on survival prevent the nerve cell and the use of these active substances for the prevention or treatment of a TSE.
Derartige Wirksubstanzen sind beispielsweise Substanzen, welche die Bindung von abnormalem Prion-Protein an eine Nervenzelle, im besonderen an ein Motoneuron, verhindern können, oder endogene Proteasen stimulieren, oder das Targeting von PrPc in Caveoli-Domainen verhindern oder die Signalkaskade, aktiviert durch Prpc nach Bindung von Prionen modulieren.such Active substances are, for example, substances that bind from abnormal prion protein to a nerve cell, in particular to a motor neuron, can prevent or stimulate endogenous proteases, or targeting PrPc in Prevent Caveoli domains or the signal cascade, activated by Modulate Prpc after binding prions.
Zu diesen Wirksubstanzen gehören beispielsweise: Antikörper oder ein Fragment eines Antikörpers, spezifisch für Prpc, Antikörper oder ein Fragment eines Antikörpers spezifisch für ein abnormales Prion-Protein, beispielsweise für PrPSC oder für PrPCJD, Peptide, welche die Bindung des abnormalen Prion-Proteins mit einer Nervenzelle, beispielsweise dem Prpc, inhibieren, Peptidomimetika, welche die Bindung von abnormalem Prion-Protein an an eine Nervenzelle, beispielsweise an Prpc verhindern können, Nukleinsäuren kodierend für einen Antikörper, eine Antikörperfragment oder ein Peptid, welche für eine Aminosäuresequenz kodiert, welche die Bindung des abnormalen Prion-Proteins an eine Nervenzelle, beispielsweise an PrPc verhindert, Proteasen, die das PrPSC abbauen, Substanzen, die die Translokation von PrPC in Caveoli inhibieren.These active substances include, for example: antibodies or a fragment of an antibody, specific for Prpc, antibodies or a Fragment of an antibody specific for an abnormal prion protein, for example for PrPSC or for PrPCJD, peptides which inhibit the binding of the abnormal prion protein with a nerve cell, for example the Prpc, peptidomimetics which bind to the abnormal prion protein can prevent a nerve cell, for example from Prpc, nucleic acids coding for an antibody, an antibody fragment or a peptide which codes for an amino acid sequence which prevents the binding of the abnormal prion protein to a nerve cell, for example to PrPc, proteases which degrade the PrPSC , Substances that inhibit the translocation of PrPC in Caveoli.
Derartige Wirksubstanzen werden in einer dem Fachmann hinlänglich bekannten Zubereitung zur parenteralen, lokalen. aerogenen oder auch oralen Verabreichung einem Patienten oder einer Person, welche im Verdacht steht, mit Prionen infiziert worden zu sein, in den Blutkreislauf, in eine Körperhöhle, in das Bindegewebe, in ein Organ, besonders intraventrikulär oder subarachnoidal in das ZNS, nasal oder bronchial verabreicht. Die Verabreichung erfolgt in der minimal effektiven Dosis jedoch nicht über der maximal tolerablen Dosis der jeweiligen Wirksubstanz mindestens einmalig baldmöglichst nach einer Infektion oder auch mehrfach, täglich oder wöchentlich über einen Zeitraum von bevorzugterweise zwei Wochen bis 6 Monaten.such Active substances are used in a preparation which is well known to the person skilled in the art parenteral, local. aerogenic or oral administration a patient or a person suspected of having Prions have been infected in the bloodstream, in a Body cavity, in the connective tissue, in an organ, especially intraventricular or subarachnoid administered to the CNS, nasally or bronchially. The administration however, the minimal effective dose does not exceed the maximum tolerable dose of the respective active substance at least once as soon as possible after an infection or several times a day or weekly over one Period of preferably two weeks to 6 months.
Beispiele zur Erläuterung der Erfindung.Examples for explanation the invention.
Beispiel 1: Herstellung von Motoneuronenkulturen vom spinalen Rückenmark der embryonalen Maus.Example 1: Production of motor neuron cultures from the spinal cord of the embryonic mouse.
Eingesetzt
wurden die folgenden Materialien:
2-Mercaptoethanol (Sigma,
Taufkirchen, Germany), B27 (Invitrogen, Karlsruhe, Germany), Brain
derived neurotrophic factor (BDNF; Cell Ceoncepts, Umkirch, Germany),
Ciliary neurotrophic factor (CNTF, kind gift, M.Sendtner), Depolarizing
saline (0.8% NaCl, 35 mM KCl and 1 μM 2-mercaptoethanol), Falcon
15 ml tubes (Sarstedt, Nürmbrecht,
Germany), Falcon 50 ml tubes (Sarstedt, Nürmbrecht, Germany), Glas cover
slips (Hartenstein, Würzburg,
Germany), Glutamax I (Invitrogen, Karlsruhe, Germany) goat serum (Invitrogen,
Karlsruhe, Germany), Greiner 4-well dishes (Greiner, Nürtingen,
Germany), Hanks balanced salt solution (HBSS w/o Ca2+, Mg2+, Invitrogen, Karlsruhe,
Germany), HEPES (Invitrogen, Karlsruhe, Germany), Horse serum (Linaris,
Wertheim, Germany), KCl (p.A., Merck, Darmstadt, Germany), Laminin-1
(Boehringer/Roche, Mannheim, Germany), Merosin (Laminin-2) (Sigma,
Taufkirchen, Germany), Na-Borat (Merck, Darmstadt, Germany), NaCl
(p.A., Merck, Darmstadt, Germany), Neurobasal (Invitrogen, Karlsruhe,
Germany), Nunclon 10cm petri dishes (cell culture grade; Brandt,
Germany), Nunclon 24-well dishes, (Brandt, Heidelberg, Germany),
Nunclon 3 cm petri dishes (cell culture grade; Brandt, Germany),
Poly-DL-Ornithine (Sigma, Taufkirchen, Germany), Rat anti mouse
p75 antibody (Chemicon, Hofheim, Germany), Tris Base (pA, Merck,
Darmstadt, Germany), Trypsin (Worthington, New Jersey MA, USA via
Cell Systems, St. Katharinen, Germany), Trypsin-Inhibitor from soy
bean (Sigma, Taufkirchen, Germany), 1 % (w/v) Gehirnhomogenat einer Maus
(C57B1/6), die mit Prionen bzw. Puffer allein behandelt worden ist.The following materials were used:
2-Mercaptoethanol (Sigma, Taufkirchen, Germany), B27 (Invitrogen, Karlsruhe, Germany), Brain derived neurotrophic factor (BDNF; Cell Ceoncepts, Umkirch, Germany), Ciliary neurotrophic factor (CNTF, kind gift, M.Sendtner), depolarizing saline (0.8% NaCl, 35 mM KCl and 1 μM 2-mercaptoethanol), Falcon 15 ml tubes (Sarstedt, Nürmbrecht, Germany), Falcon 50 ml tubes (Sarstedt, Nürmbrecht, Germany), glass cover slips (Hartenstein, Würzburg, Germany ), Glutamax I (Invitrogen, Karlsruhe, Germany) goat serum (Invitrogen, Karlsruhe, Germany), Greiner 4-well dishes (Greiner, Nürtingen, Germany), Hanks balanced salt solution (HBSS w / o Ca2 +, Mg2 +, Invitrogen, Karlsruhe , Germany), HEPES (Invitrogen, Karlsruhe, Germany), Horse serum (Linaris, Wertheim, Germany), KCl (pA, Merck, Darmstadt, Germany), Laminin-1 (Boehringer / Roche, Mannheim, Germany), Merosin (Laminin -2) (Sigma, Taufkirchen, Germany), Na-Borat (Merck, Darmstadt, Germany), NaCl (pA, Merck, Darmstadt, Germany), Neurobasal (Invitrogen, Ka rlsruhe, Germany), Nunclon 10cm petri dishes (cell culture grade; Brandt, Germany), Nunclon 24-well dishes, (Brandt, Heidelberg, Germany), Nunclon 3 cm petri dishes (cell culture grade; Brandt, Germany), Poly-DL-Ornithine (Sigma, Taufkirchen, Germany), Rat anti mouse p75 antibody (Chemicon, Hofheim, Germany), Tris Base (pA, Merck, Darmstadt, Germany), Trypsin (Worthington, New Jersey MA, USA via Cell Systems, St. Katharinen, Germany), Trypsin-Inhibitor from soy bean (Sigma , Taufkirchen, Germany), 1% (w / v) brain homogenate of a mouse (C57B1 / 6) that was treated with prions or buffer alone.
Die Kultivierung embryonaler Motoneuronen der Maus wurde wie folgt durchgeführt. Kulturen von embryonalen spinalen Motoneuronen der Maus (Alter der Embryonen 12.5 Tage) werden mit Hilfe der Panning-Methode unter Verwendung eines Ratte anti Maus p75 monoklonalen Antikörpers (Chemicon, Hofheim) durchgeführt (Wiese et al., 1999; Wiese et al., 2001). Die Embryonen werden dem Uterus entnommen und mikrodissektiert unter dem Mikroskop. Die ventrolateralen Teile des lumbalen Rückenmarks werden in HBSS mit 10μM 2-Mercaptoethanol transferiert, anschliessend für 10 min mit 0,1 % Trypsin (in HBSS) bei 37°C behandelt und schliesslich trituriert und mit 0.1 % Trypsin-Inhibitor (fin. conc) behandelt. Die vereinzelten Zellen werden auf eine mit p75-Antikörper (10ng/ml in 10mM Tris/HCl pH 9,5) beschichtete Zellkulturplatte (24 well Platte bei Einzelembryopräparationen, 10 cm Platte im Falle der Gesamtembryo-Präparationen) gegeben und das Panning für 30 min bei Raumtemperatur durchgeführt. Anschliessend wurden die einzelnen wells/die Zellkulturplatte jeweils 3x gewaschen mit HBSS und die Motoneuronen mit Depolarisierungslösung (0,8 % NaCl, 35 mM KCl) abgelöst. Die Suspension wird mit 1:1 Motoneuronenmedium (Neurobasal, 1x B27, 1% Glutamax, 10% Pferdeserum supplementiert, bei 400g 5 min zentrifugiert und das Zellsediment in Motoneuronenmedium resuspendiert. Die Zellzahl wird bestimmt und die Zellen auf mit Poly-DL-Ornithine und Laminin-1 oder Poly-DL-Ornithine und Merosin beschichtete Platten in einer Dichte von 2000 Zellen/cm2 plattiert. Im Falle der Behandlung der Zellen mit Gehirnextrakt von Prpsc-infizierten und MOCK-infizierten Mäusen wurde ein 1% Gehirnhomogenat in HBSS hergestellt. Von diesem Gehirnhomogenat wurden jeweils eine 1:103 bis 1:107 (in 10er Potenzschritten abwärts) als Verdünnung (in HBSS) zur abschliessenden Beschichtung der Platten verwendet. Die Beschichtung wurde für 30 min bei Raumtemperatur durchgeführt. Die Motoneuronen wurden mit BDNF, GDNF oder CNTF als neurotrophen Faktor behandelt (jeweils in 10 ng/ml Endkonzentration im Motoneuronenmedium). Ein Mediumwechsel wurde an Tag 1 und 3 und 5 durchgeführt. Die Motoneuronen wurden an Tag 0 drei Stunden nach dem Plattieren gezählt und dann an Tag 1, 3 und 5. Es wurden jeweils 10 Felder (1.16 mm2/Feld) gezählt.The cultivation of mouse embryonic motor neurons was carried out as follows. Cultures of mouse embryonic spinal motor neurons (age of embryos 12.5 days) are carried out using the panning method using a rat anti mouse p75 monoclonal antibody (Chemicon, Hofheim) (Wiese et al., 1999; Wiese et al., 2001 ). The embryos are removed from the uterus and microdissected under the microscope. The ventrolateral parts of the lumbar spinal cord are transferred to HBSS with 10μM 2-mercaptoethanol, then treated for 10 min with 0.1% trypsin (in HBSS) at 37 ° C and finally triturated and with 0.1% trypsin inhibitor (fin. Conc) treated. The isolated cells are placed on a cell culture plate coated with p75 antibody (10ng / ml in 10mM Tris / HCl pH 9.5) (24 well plate for single embryo preparations, 10 cm plate in the case of total embryo preparations) and the panning for 30 min carried out at room temperature. The individual wells / the cell culture plate were then washed 3 times with HBSS and the motor neurons were detached with depolarizing solution (0.8% NaCl, 35 mM KCl). The suspension is supplemented with 1: 1 motor neuron medium (Neurobasal, 1x B27, 1% Glutamax, 10% horse serum, centrifuged at 400 g for 5 min and the cell sediment is resuspended in motor neuron medium. The cell number is determined and the cells are opened with poly-DL-ornithine and Laminin-1 or Poly-DL-Ornithine and Merosin coated plates plated at a density of 2000 cells / cm 2. In the case of treating the cells with brain extract from Prpsc-infected and MOCK-infected mice, a 1% brain homogenate was produced in HBSS a 1:10 3 to 1:10 7 (in steps of 10 down) was used as a dilution (in HBSS) for the final coating of the plates, the coating was carried out for 30 min at room temperature and the motor neurons were treated with BDNF, GDNF or treated with CNTF as a neurotrophic factor (each in 10 ng / ml final concentration in the motor neuron medium.) A medium change was carried out on days 1 and 3 and 5. otoneurons were counted three hours after plating on day 0 and then on days 1, 3 and 5. 10 fields (1.16 mm 2 / field) were counted in each case.
Beispiel 2: Immunhistochemie zur Detektion von Axon und DendritenExample 2: Immunohistochemistry for the detection of axons and dendrites
Es wurden die folgenden Materialien eingesetzt: 85% Glycerin solution (Merck, Darmstadt, Germany), Mowiol 40-88 (Sigma, Taufkirchen, Germany), Paraformaldehyde (Merck, Darmstadt, Germany), 100 mM Phosphate buffered saline (w/o Ca2+, Mg2+, PBS, pH 7.5; Invitrogen, Karlsruhe, Germany), Goat serum (Invitrogen, Karlsruhe, Germany), Tris buffered saline (TBS), 100 mM Tris, pH 7.5, 0.7 % NaCl), Triton-x-100 (Sigma, Taufkirchen, Germany), Rabbit anti mouse Tau antibody (Sigma, Taufkirchen, Germany), Mouse anti human MAP-2 antibody (Chemicon, Hofheim, Germany), goat anti rabbit Cy3 (Dianova, Hamburg, Germany), rabbit anti mouse Cy2 (Dianova, Hamburg, Germany).It the following materials were used: 85% glycerol solution (Merck, Darmstadt, Germany), Mowiol 40-88 (Sigma, Taufkirchen, Germany), Paraformaldehyde (Merck, Darmstadt, Germany), 100 mM phosphate buffered saline (w / o Ca2 +, Mg2 +, PBS, pH 7.5; Invitrogen, Karlsruhe, Germany), Goat serum (Invitrogen, Karlsruhe, Germany), Tris buffered saline (TBS), 100 mM Tris, pH 7.5, 0.7% NaCl), Triton-x-100 (Sigma, Taufkirchen, Germany), Rabbit anti mouse Tau antibody (Sigma, Taufkirchen, Germany), Mouse anti human MAP-2 antibody (Chemicon, Hofheim, Germany), goat anti rabbit Cy3 (Dianova, Hamburg, Germany), rabbit anti mouse Cy2 (Dianova, Hamburg, Germany).
Die kultiverten Motoneuronen wurden mit 4% Paraformaldehyd in 100 mM PBS fixiert (30 min Raumtemperatur). Die Zellen wurden 3x gewaschen mit TBS (Tris gepufferte Saline, 100 mM Tris, pH 7.5, 0.7 % NaCl, je 10 min bei 20°C bzw. Raumtemperatur) und anschliessend einem Blocking-Schritt unterworfen mit TBS + 10 % Ziegenserum + 0,05 % Triton-x-100 für 30 min bei Raumtemperatur. Die Blocking-Lösung wurde anschliessend ausgetauscht gegen eine Blocking-Lösung, die die Antikörper gegen Tau (polyklonal, Sigma, Deisenhofen, 1:400) und MAP-2 (monoklonal Maus anti Map-2; 5 μg/ml). Die Zellen wurden über Nacht bei 4°C mit dem Antikörpergemisch inkubiert. Anschliessend wird 3x gewaschen mit TBS und erneut blockiert, wie oben angegeben. Dann werden die Zellen mit je 2 μg/ml Ziege anti Kaninchen Cy3 und Kaninchen anti Maus Cy2 in TBS + 10% Ziegenserum + 0,05 Triton-x-100 inkubiert für 30 min bei Raumtemperatur. Die Zellen werden dann gewaschen mit TBS (3x je 10 min bei Raumtemperatur) und die Deckgläschen mit Hilfe von Mowiol (10g in 40 ml PBS) in 50 % Glycerin eingedeckelt.The cultivated motor neurons were treated with 4% paraformaldehyde in 100 mM PBS fixed (30 min room temperature). The cells were washed 3 times with TBS (Tris buffered saline, 100 mM Tris, pH 7.5, 0.7% NaCl, each 10 min at 20 ° C or room temperature) and then subjected to a blocking step with TBS + 10% goat serum + 0.05% Triton-x-100 for 30 min at room temperature. The blocking solution was then replaced against a blocking solution, which are the antibodies against dew (polyclonal, Sigma, Deisenhofen, 1: 400) and MAP-2 (monoclonal Mouse anti Map-2; 5 μg / ml). The cells were over Night at 4 ° C with the antibody mixture incubated. It is then washed 3 times with TBS and blocked again, as you can read above. Then the cells with 2 μg / ml goat anti Rabbit Cy3 and rabbit anti mouse Cy2 in TBS + 10% goat serum + 0.05 Triton-x-100 incubated for 30 min at room temperature. The cells are then washed with TBS (3x 10 min each at room temperature) and the coverslips with Covered with Mowiol (10g in 40ml PBS) in 50% glycerin.
Die Bestimmung der Axon- und Dendritenlängen wurde wie folgt durchgeführt. Bilder der immungefärbten Motoneuronen wurden mit Hilfe eines konfokalen Mikroskops (Leica TCS, Heidelberg, Germany) aufgenommen. Axonale Fortsätze konnten anhand der Cy3 (rot) Färbung von dentritischen Fortsätzen (Cy2, grün) unterschieden werden. Die Fortsätze wurden mit Hilfe des Programms Scion Image vermessen und die Daten zur weiteren Analyse in GraphPad Prizm analysiert.The Determination of the axon and dendrite lengths was carried out as follows. pictures the immunostained Motor neurons were detected using a confocal microscope (Leica TCS, Heidelberg, Germany). Axonal processes could be based on the Cy3 (red) coloring of dendritic processes (Cy2, green) be distinguished. The extensions were measured using the Scion Image program and the data Analyzed in GraphPad Prizm for further analysis.
Beispiel 3: Herstellung des Prpsc und MOLK-Gehirnextrakts.Example 3: Production of the Prpsc and MOLK brain extract.
RML (Rocky Mountain Laborarory), ein Maus-adaptiertes Scrapie Isolat (Chandler, 1961), wurde passagiert in CD-1 Mäusen (Charles River Laboratories). Inocula Stocks waren 10% (w/v) Homogenate von RML-infizierten, terminal erkrankten CD1 Maus-Gehirnen in 0.32 M Sucrose. Mäuse wurden behandelt mit 30 μl i.c. einer 10-fachen Verdünnung des Stock in HBSS mit 5% Rinderserum Albumin (BSA). Die Infektiösitätstiter wurden mittels Infektion von PrP überexprimierenden tga20/tga20 Mäusen (Fischer et al., 1996) mit 30 μl des verdünnten Homogenats injiziert in die rechte parietalen Gehirnhälfte bestimmt. Prionen Titer wurden berechnet anhand der Zeit, die die infizierten Mäuse bis zur terminalen Erkrankung benötigten. (Prusiner et al., 1982; Brandner et al., 1996). MOCK Extrakt wurde in Parallel-Ansätzen mit Homogenat von nicht infizierten Gehirnen hergestellt.RML (Rocky Mountain Laborarory), a mouse-adapted scrapie isolate (Chandler, 1961) was passaged in CD-1 mice (Charles River Laboratories). Inocula stocks were 10% (w / v) homogenates of RML-infected terminal diseased CD1 mouse brains in 0.32 M sucrose. Mice were treated with 30 μl i.c. a 10-fold dilution of the Stock in HBSS with 5% bovine serum albumin (BSA). The infectiousness titre were overexpressing tga20 / tga20 by infection of PrP Mice (Fischer et al., 1996) with 30 ul the diluted Homogenates injected into the right parietal hemisphere. Prion titers were calculated based on the time it took for the infected Mice up needed for terminal illness. (Prusiner et al., 1982; Brandner et al., 1996). MOCK extract was in parallel approaches made with homogenate from non-infected brains.
Beispiel 4: Experimente und Ergebnisse zur Einfluss von abnormalen Prionen auf Axone und Überlebensraten.Example 4: Experiments and results on the influence of abnormal prions on axons and survival rates.
Es wurden in unterschiedlichen Experimenten zunächst Motoneuronen des lumbalen Rückenmarks in Einzelembryo-Präparationen aus Verpaarungen von Prnp +/– X Prnp +/– (auf C57B1/6/129SV Hintergrund gehalten) isoliert (Bueler et al., 1992). Die Genotypisierung der Embryonen erfolgte über DNA-Extraktion aus dem Restgewebe der präparierten Embryonen und die Genotypisierung erfolgte entsprechend der Angaben von Büeler H, Aguzzi A, Sailer A, et al. 1993 "Mice devoid of PrP are resistant to scrapie" Cell 73:1339-47. Die auf Poly-DL-Ornithine und Laminin-1 bzw. Poly-DL-Ornithine und Merosin kultivierten Motoneuronen wurden an Tag 0, 1, 3 und 5 gezählt und an Tag 7 fixiert für die nachfolgenden Färbungen gegen MAP-2 und Tau (Dendriten bzw. Axon-spezifisch (Bommel et al., 2002). Anschliessend wurden die Axonlängen und die Dendritenlängen vermessen mit Hilfe des Computerprogramms Scion Image.It In different experiments, lumbar motor neurons were initially identified Backmarks in single embryo preparations from matings of Prnp +/– X Prnp +/– (on C57B1 / 6 / 129SV background)) isolated (Bueler et al., 1992). The The embryos were genotyped by DNA extraction from the Residual tissue of the prepared Embryos and genotyping were carried out according to the information by Büeler H, Aguzzi A, Sailer A, et al. 1993 "Mice devoid of PrP are resistant to scrapie" Cell 73: 1339-47. Those on Poly-DL-Ornithine and Laminin-1 or Poly-DL-Ornithine and Merosine-cultured motor neurons were counted on day 0, 1, and 5 and fixed on day 7 for the following colors against MAP-2 and tau (dendrites or axon-specific (Bommel et al., 2002). The axon lengths and the dendrite lengths were then measured with the help of the computer program Scion Image.
In der weiteren Abfolge wurden lumbale Motoneuronen von CD-1 Mausembryonen isoliert und kultiviert. Die Plattenbeschichtung erfolgte, wie oben bereits angegeben, zunächst mit Poly-DL-Ornithine, Laminin-1 und anschliessend Gehirnextrakt von RML-infizierten Mäusen bzw. MOCK infizierten Mäusen. Die Gehirnextrakte wurden in unterschiedlichen Konzentrationen auf die beschichteten Platten gegeben (s.u.). Die kultivierten Motoneuronen wurden an Tag 0, 1, 3 und 5 gezählt und an Tag 7 fixiert für die nachfolgenden Färbungen gegen MAP-2 und Tau (Dendriten bzw. Axon-spezifisch). Anschliessend wurden die Axonlängen und die Dendritenlängen vermessen mit Hilfe des Computerprogramms Scion Image.In the further sequence were lumbar motor neurons from CD-1 mouse embryos isolated and cultivated. The plate was coated as above specified, initially with poly-DL-ornithine, laminin-1 and then brain extract of RML-infected mice or MOCK infected mice. The brain extracts were found in different concentrations given the coated panels (see below). The cultivated motor neurons were counted on days 0, 1, 3 and 5 and fixed on day 7 for the following colors against MAP-2 and tau (dendrites or axon-specific). Then were the axon lengths and the dendrite lengths measured with the help of the computer program Scion Image.
Es wurden die folgenden Ergebnisse erhalten. Die Überlebensraten für kultivierte lumbale Prnp ko Motoneuronen im Vergleich zu Prnp +/– oder Prnp +/+ (wt) zeigten keine signifikanten Unterschiede im Überleben dieser Motoneuronen. Die Vermessung von Dendritenlängen der kultivierten lumbalen Prnp ko Motoneuronen im Vergleich zu Prnp +/+ (wt) zeigten keine signifikanten Unterschiede in den Dendritenlängen dieser Motoneuronen. Die Vermessung von Axonlängen der kultivierten lumbalen Prnp ko Motoneuronen im Vergleich zu Prnp +/+ (wt) zeigten signifikante Unterschiede in den Axonlängen dieser Motoneuronen sowohl auf mit Poly-DL-Ornithine und Laminin-1 als auch auf mit Poly-DL-Ornithine und Merosin beschichteten Deckgläschen.The following results were obtained. The survival rates for cultured lumbar Prnp ko motor neurons compared to Prnp +/- or Prnp + / + (wt) showed no significant differences in the survival of these motor neurons. The Measurement The dendrite lengths of the cultured lumbar Prnp ko motor neurons compared to Prnp + / + (wt) showed no significant differences in the dendrite lengths of these motor neurons. The measurement of axon lengths of the cultured lumbar Prnp ko motor neurons compared to Prnp + / + (wt) showed significant differences in the axon lengths of these motor neurons both with Poly-DL-Ornithine and Laminin-1 as well as with Poly-DL-Ornithine and Merosin coated coverslips.
Die Überlebensraten für kultivierte lumbale embryonale Motoneuronen vom Wildtyp, die auf mit Poly-DL-Ornithine und Laminin-1, sowie entweder mit Prionen oder MOCK beschichteten Deckgläschen kultiviert wurden, unterschieden sich signifikant in einem Bereich von 1:1000 bis 1:1000.000, d.h. in einem Bereich von 0,1% bis 0,001 (Endkonzentration bei der Beschichtung) verwendeten Gehirnextrakt. Der Prionen Gehirnextrakt führte zu einem signifikant geringeren Überleben von kultivierten lumbalen embryonalen Motoneuronen in Kultur bei einer Konzentration des prionhaltigen Hirnextrakts von 1:1.000 und 1:10.000 im Vergleich zu Motoneuronen, die in Gegenwart von MOCK-Extrakt kultiviert wurden. Die Dendritenlängen wurden bei einer Prionen bzw. MOCK-Extrakt-Endkonzentration bei der Beschichtung von 0,1% bestimmt und unterschieden sich nicht signifikant voneinander. Die Axonlängen wurden bei einer Prionen-Hirnextrakt bzw. MOCK-Extrakt- Endkonzentration bei der Beschichtung von 0,1 % bestimmt und unterschieden sich signifikant voneinander. Der Prionen Gehirnextrakt führte zu signifikant geringeren Axonlängen von kultivierten lumbalen embryonalen Motoneuronen in Kultur im Vergleich zu Motoneuronen, die in Gegenwart von MOCK-Extrakt kultiviert wurden.The survival rates for cultivated lumbar wild-type embryonic motor neurons that are coated with poly-DL-ornithine and Laminin-1, as well as coated with either prions or MOCK cover slip cultivated differed significantly in one area from 1: 1000 to 1: 1000,000, i.e. in a range of 0.1% to 0.001 (Final concentration in the coating) used brain extract. The Prions brain extract resulted to significantly lower survival of cultured lumbar embryonic motor neurons in culture a concentration of the prion-containing brain extract of 1: 1,000 and 1: 10,000 compared to motor neurons cultured in the presence of MOCK extract were. The dendrite lengths were at a prion or MOCK extract final concentration the coating of 0.1% determined and did not differ significantly from each other. The axon lengths were at a prion brain extract or MOCK extract final concentration determined at the coating of 0.1% and differed significantly from each other. The prion brain extract resulted in significantly less Axonlängen of cultivated lumbar embryonic motor neurons in culture in the Compared to motor neurons cultivated in the presence of MOCK extract were.
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DE2003113098 DE10313098A1 (en) | 2003-03-24 | 2003-03-24 | Test system for finding active substances against prion-induced diseases and active substances for the prevention and treatment of this disease |
DE112004000945T DE112004000945D2 (en) | 2003-03-24 | 2004-03-24 | Test system for the discovery of drugs against prion-induced diseases and drugs for the prevention and treatment of these diseases |
PCT/DE2004/000645 WO2004086051A1 (en) | 2003-03-24 | 2004-03-24 | Test system for discovering active ingredients against prion-induced illnesses and active ingredients for preventing and treating said illnesses |
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Büeler, H. u.a.: Mice devoid of PrP are resistant to scrapie. Cell (1993) 73(7)1339-47 * |
Chen, S. u.a.: Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival. Mol. Cell. Neurosci. (Feb. 2003) 22(2)227-33 * |
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