DE10339727A1 - Method for the determination of adult neurogenesis - Google Patents

Abstract

The invention relates to a method for the determination of adult neurogenesis, the use of transiently postmitotically expressed endogenous cell constituents for the determination of adult neurogenesis and a bioassay and a kit for the determination of these.

Description

The The invention relates to a method for the determination of adult neurogenesis, the use of transient postmitotic expressed endogenous Cell components for the determination of adult neurogenesis and a Bioassay and a kit to determine this.

The modern neurobiology is over shaped large parts of the view, that nothing can regenerate in an adult brain. These Conception has appeared in textbooks so far set that can be spoken of a dogma. In recent years, observations are made again and again which can not be reconciled with this dogma. For example, it was found in brain injuries that by modifications of the structure in non-injured areas partly tasks of the destroyed ones Areas taken over This modification is an indication of plasticity in the adult Brain were.

A Neurogenesis in the adult brain was first established in 1965 by Altman and This is described as adult hippocampal neurogenesis. These and then im Further detailed experimental data contradicted the common Presentation of the architecture of the brain and the extent of the possible Plasticity. In particular, through the work of Notteboom could be shown that in songbirds the process of learning with a massive neoplasm of nerve cells correlated. However, since first an adult hippocampal neurogenesis was not found in primates However, the relevance of these studies in biomedical, anthropocentric view questionable. In 1996 Kuhn et al made experimental experimental arrangements be established with whom the adult hippocampal neurogenesis in the monkey and finally could also be described in humans.

In the subgranular Zone of the dentate gyrus exists a population dividing Parent or progenitor cells. Daughter cells resulting from asymmetric divisions of these cells, suggest the way to differentiation into nerve or glial cells one. It survives only a fraction of the newborn cell, mostly the majority is about apoptotic Mechanisms eliminated. As in embryogenesis, so too in adult neurogenesis a surplus to be produced by potential nerve cells, by which selection processes the really needed Cells are recruited, the processes of cell birth and the Cell deaths closely linked are.

Lots Neurological disorders are associated with physiological and pathophysiological Operations. the modulation of adult neurogenesis or accompanying phenomena connected or have a component that is the brain region of the hippocampus concerns. In particular, learning disorders and memory disorders the consequence of such changes. The most important diseases in this context are Alzheimer's disease, Temporal lobe epilepsy and endogenous depression. It will suspected that these diseases are a chronic disorder of the adaptability underlying the hippocampus. Because the hippocampus - as already executed - different as other brain regions can produce new neurons for life, concerns the disturbed plasticity also the formation of neurons from local stem and progenitor cells. It is therefore agreed in the professional world that drugs that may modulate adult neurogenesis, promising candidates for novel Treatment options for these diseases are.

Disadvantageously However, adult neurogenesis has so far been very difficult to detect and to determine. In the known methods is an injection a marker substance is necessary, which marks the division active cells. After the marking, it is possible to wait until after a certain time, which allows the new cells to mature, neurogenesis immunohistochemically be examined by the distribution of markers. For example, substances to investigate their effect on adult neurogenesis in vivo, needed Therefore, a high number of experimental animals, a complex experimental Design and a long trial period.

task The present invention is therefore a method and a To provide an assay that allows adult neurogenesis to be simple, safe and secure can be determined effectively.

The invention solves this problem by a method for the determination of adult neurogenesis, wherein in at least one nerve cell transiently postmitotically expressed endogenous cell constituents are determined. For the purposes of the invention, it may be provided, for example, to determine the adult neurogenesis in the absence and presence of substances which are to be examined as to whether their presence or absence alters or modulates adult neurogenesis. For this purpose, the substance to be tested for its neurogenic activity can be administered, for example, to a test animal in a manner standardized for the test series in question and for a specific duration. This can be preferred by daily parenteral administration of the substance for a period of 1 to 4 weeks. The exact values are known to the person skilled in the art and are based on previous experience and, if appropriate, pilot tests with the corresponding substances. At the end of the particular experimental time determined by pilot experiments, the test organisms are sampled from the brain or the brain completely, preferably they are killed in advance. Of course, it can also be provided to remove only a specific area of the brain so that the killing of the experimental organism is not necessarily in accordance with the invention. The removed brain or the extracted tissue parts can be fixed and prepared according to the usual standard procedures for the histological or for the immunohistological examination. With specific recognition substances, the transiently postmitotically expressed cell components and thus the immature nerve cells are visualized in the histological or in the immunohistochemical procedure. The recognition substances may be, for example, substances which can detect the cell components at the nucleic acid or at the amino acid level. These may be, for example, antibodies, fluorescent marker substances, antisense constructs, chelators or other structures known to those skilled in the art, which may interact with cell components preferably at the protein level such that their detection and in particular their qualitative and quantitative determination are possible is. The endogenous cell constituents to be detected may be, for example, expressed proteins or carbohydrates and lipids. Under transient postmitotic expression in the. The invention relates to the measurability of the selected endogenous cell constituent during a maturation period of a new nerve cell after completion of cell division, which is distinctively above or below the corresponding measurability in mature nerve cells.

If a cell component only temporary and only during a particular stage of development of the cell is expressed, so lets Conversely, by detection of this cell component in experience Bring if a particular cell at the time of examination at this stage of development.

The Interaction of the recognition molecules with those to be detected endogenous cell constituents to mark particular cells whose number is determined and optionally to the total number of nerve cells in the relevant brain region in proportion is set. The effectiveness of different substances on the production of young Nerve cells can be compared so easily, safely and efficiently and as a measure of nerve cell formation in the adult brain and the neurogenic efficacy of the studied Substances are taken. A substance or another measure, leading to more nerve cells at an early stage of development leads, would as a substance found in the presence of only a few such cells are considered superior in this regard become.

It is self-evident possible, the method with a marker or with a combination of two or more. So it can be provided, a percentage of certain To use markers. The procedure can be invasive or non-invasive carried out become; Of course Is it possible, to combine a non-invasive and an invasive procedure. So can first one Determination using one or more markers non-invasively, following an invasive determination with the same or other markers as in the non-invasive determination is performed.

One this method allows a quick implementation the testing of adult neurogenesis and there will be more information obtained from a single experiment, with a sequence of experiments, but also a single experiment with the method according to the invention easily standardized. If the procedure on experimental animals carried out will result in a minimization of the experimental animals to be used. It is within the meaning of the invention, of course, not mandatory that the procedure was performed on animals It will, of course, be also be applied in the human area. If the inventive method applied to humans, the qualitative and / or quantitative determination of adult neurogenesis preferred by the acquisition of newly formed cells, using noninvasive procedures and devices are used. This can be for example to act in imaging and nuclear medicine procedures, such as Positron emission tomography, SPECT, computed tomography and / or magnetic resonance imaging and other.

Of the Detection of adult neurogenesis by means of said markers, in particular Calretinin has several advantages over the known methods.

In order to make statements about the effectiveness of a substance in the regulation of nerve cell formation, purely qualitative statements are unhelpful, since, if necessary, the superiority of one substance should be documented over another and this just in a countable and measurable extra nerve cells expresses.

In Certain experimental contexts can also mean that advantages in terms of purely qualitative statements. Especially calretinin is expressed in the cytoplasm of the new nerve cells, so that z. B. immunohistochemical labeling of calretinin the structure makes the cell clearly visible. If an active substance to be tested is This can be easily detected on the appearance of the cell. Again, this is the known methods whose marker antigens themselves only in the nucleus, differently: while the cell body and his extensions a changed one Making morphology highly visible makes it much more difficult at the nucleus read. So, especially with calretinin, the newly formed one becomes Make nerve cells easier to see, whether they have developed normal and healthy.

• • Hohe Sensitivität und Spezifität
• • Einfache, direkte Quantifizierbarkeit mit hoher Genauigkeit und Verlässlichkeit
• • Qualitative Aussagen zur Zellstruktur und Morphologie sind problemlos im gleichen Untersuchungsgang möglich
• • Geringerer experimenteller Aufwand mit weniger Tieren und billigeren Reagenzien
• • potentiell gute Automatisierbarkeit
• • Einfacherer Aufbau des Tests (nur eine histologische Färbung, die am normalen Lichtmikroskop ausgewertet werden kann und ebenfalls potentiell automatisierbar ist)
• • Die Versuchsorganismen erhalten einzig die Testsubstanz, deren Wirkung auf die Nervenzellneubildung untersucht werden soll, aber keine zusätzliche Markersubstanz, so dass die Auswirkungen möglicher Interaktionen zweier applizierter Substanzen auf das Messergebnis entfallen.

The method of estimating the number of newly formed nerve cells by the number of cells expressing calretinin has significant practical advantages. All new nerve cells have a so-called calretinin stage, but this stage does not follow until the number of cells changes no more, since the cells in the calretinin stage have stopped cell division activity, ie have become postmitotic. Therefore, the method of the invention is very sensitive. Since only the newly formed nerve cells express calretinin or the other named markers, the method according to the invention is also very specific. The main advantage of the new method is that, especially with calretinin, there is a marker that distinguishes the newly formed cells from other cells in the same brain region so that they can be easily detected. In any other known method, the developing cells must first be elaborately labeled in order to be able to determine, post hoc, in a further step, whether labeled cells have developed into new nerve cells. This is more cumbersome and less economical, requires more animals in animal experiments, if invasive procedures are carried out with laboratory animals, increases the probability of error, since the evaluation is more difficult, and is also hardly automatable, quantitatively, the classical method is somewhat less accurate, since the analysis in Two steps must be taken. The advantages of the Calretinin method are thus:

• • High sensitivity and specificity
• • Simple, direct quantifiability with high accuracy and reliability
• • Qualitative statements on cell structure and morphology are easily possible in the same examination
• • Less experimentation with fewer animals and cheaper reagents
• • potentially good automation
• • simpler structure of the test (only a histological staining, which can be evaluated on the normal light microscope and is also potentially automatable)
• • The test organisms receive only the test substance, whose effect on the nerve cell formation is to be examined, but no additional marker substance, so that the effects of possible interactions of two applied substances on the measurement result omitted.

In a preferred embodiment the invention is in the postmitotic expressed endogenous cell components to proteins, carbohydrates and / or Including lipids their modifications, such as phosphorylations, glycosylations, Polysylations and enzymatic cleavages. Especially preferred are the calcium-binding proteins, in particular calbindin, parvalbumin, Calretinin, MRP8, MRP14 as well as the family of 5100 calcium-binding Proteins, especially S100B (or beta) and others, especially Calretinin is preferred.

In a particularly preferred embodiment the endogenous cell constituent is calretinin or calbindin. It is preferred by the detection of this Protein on the amino acid or at the nucleic acid level possible, To determine the adult neurogenesis quickly and safely. In addition to parvalbumin, Calbindin belongs to calretinin to the calcium-binding proteins present in different types of neurons can be detected. Calretinin is a calcium binding protein found in certain Types of nerve cells, especially interneurons is expressed. It however, is also found to be a transiently expressed molecule in ripening granule cells of the hippocampus. Calbindin is a calcium binding protein found in production, stabilization and maintenance of cytoskeletal elements is involved.

In a preferred embodiment of the invention, the nerve cells are selected from the group comprising the hippocampus (dentate gyrus), olfactory bulb (olfactory bulb, lateral ventricle walls and rostral migration current) and cerebellum, projection neurons (especially pyramidal cells) of the cortex and subcortical brain sections, Principal cells in the hippocampus (dentate gyrus, hilus, CA3, CA2 and CA1) and olfactory bulb, interneurons in the hippocampus, cerebral cortex, olfactory bulb and subcortical brain sections. The group also includes neurosecretory nerve cells of the hypothalamus, Purkinje cells of the cerebellum and sensory nerve cells of the sense organs and downstream centers. For the purposes of the invention, a neuron (a nerve cell) is any cell that is capable of action potential. The cells mentioned may be part of a sample for the method according to the invention. By analyzing the sample With the method according to the invention it is possible to generate statements about the adult neurogenesis within the sample.

A Sample in the context of the invention is the name for a by Sample taken from biological material or a subset or a small amount of such, the nature of which is chemical, biological, clinical or similar In particular, to get out of this sample findings to the adult To gain neurogenesis; For example, the sample may be a collection be from cells or a piece of a brain. The sampling or recovery takes place in particular such that the extracted subset is an average of the total Quantity corresponds. The by the investigation or from the sample determined features can the assessment of adult neurogenesis in the area covered by the sample Quantity that draws conclusions the total amount allowed become. For the investigation can the samples by mixing, dividing, separating, pre-fractionating, Crushing, addition of enzymes or markers or otherwise pretreated become. The person skilled in the art has various options for pretreatment the samples known. Of course it may also be provided that the sample is removed in this way, that it does not correspond to an average of the total amount. A Specimen are primarily biological materials that comprise structures with the forwarding or storage of information biological basis are suitable. These include, for example, the CNS or parts thereof, the hippocampus (with its subsections plus adjacent areas, especially subiculum, parasubiculum) spinal cord and the extended one Mark, including the brainstem, neocortex and subcortical Brain sections, the striatum, the cerebellum and others. It can but in certain cases continue with liquids, Cell collection or tissue, e.g. preferably granule cells include; but also to structures that nerve cells (after the above Definition and list of examples). The sample can accordingly numerous other materials include, for example, blood, lymph, cerebral fluid and many more. But it is also possible that the sample only parts the nerve cells in particular, for example, the soma or the processes (neurites, neuropil) contains.

neurons For the purposes of the invention, all nerve cells are considered highly differentiated Cells for receiving, processing and relaying nervous excitations capable are. These cells come, for example, in the central, peripheral and autonomic nervous system and in the sense organs and can be one Be subset of the sample.

at The nerve cells can be, for example, nerve cells Animals or humans act, in particular granule cells, pyramidal cells are preferred and interneurons in the hippocampus and interneurons in the olfactory bulb.

The Cells can be taken from different tissues. The central nervous system is divided according to the invention in the brain and spinal cord. The nervous system one divides into peripheral, central and autonomous nervous system. Neocortex and striatum are parts of the brain. Important regions with adult neurogenesis, the hippocampus and olfactory bulb (ventricular wall, including the underlying basal ganglia, the olfactory bulb and the intervening Migration route of the rostral migration path). But of course you can too Nerve cells from other regions where neoplasm of nerve cells will be used.

in the According to the invention nerve cells (neurons), in particular cells, which occur in the CNS and especially in the hippocampus and olfactory bulb. These neurons must but not exclusively in the CNS within the meaning of the invention.

in the According to the invention, neurons or nerve cells are in particular those derived from stem and progenitor cells of the adult brain physiologically or after stimulation emerge. These cells are found throughout the brain but form physiologically only in the hippocampus and olfactory brain new neurons. Parent or progenitor cells are undifferentiated cells capable of self-renewal and from which can emerge maturing cells of one or more types. in the This differentiation can be found between the stage of Parent or progenitor cells and the mature neurons an intermediate stage, for example identifiable by the expression of calretinin.

in the In accordance with the invention, the determination of the modulation takes place in particular of adult neurogenesis by various pharmacological substances with regard to neurological and psychiatric disorders Dementia, temporal lobe epilepsy or depression.

The Modification or change across from of adult neurogenesis A normal value can be within the meaning of the invention with all of the brain outgoing or characterized by this Diseases are associated. For example, the modification neurogenesis associated with dementia.

A dementia in the sense of the invention is the loss of acquired intellectual ability, esp special memory and personality changes, for example, as a result of a brain organic disease. Dementia in the sense of the invention continues to be the decline in cognitive performance in old age. However, dementia within the meaning of the invention is also memory and memory disorders, orientation disorders, delusional states or the loss of personality.

Also innate reductions in cognitive performance, provided they have a Disruption of the Nerve cell formation is associable in the adult brain, belong to the neuropsychiatric disorders within the meaning of the invention.

Dementia is the most common cause of long-term care needs in industrialized countries. According to the invention, the diagnosis of dementia is carried out in a differential diagnostic manner, preferably based on clinical criteria. Furthermore, (i) internal, (ii) neurological and / or (iii) psychiatric conditions can be taken into account that accompany the dementia or the dementia-related or dementia-associated diseases in the sense of the invention are listed below, it being known to those skilled in the art that in dementia, the medical, (ii) neurological and / or (iii) psychiatric conditions may dominate the clinical or medical picture of dementia or may be completely absent; the internal (ii) neurological and (iii) psychiatric clinical pictures can of course be covered by the teaching according to the invention, depending on the course of the disease.
on (i): chronic obstructive pulmonary disease, chronic heart failure, cardiac arrhythmia, recurrent asystole, hypothyroidism, hyperthyroidism, hypoparathyroidism, hyperparathyroidism, chronic uremia, liver cirrhosis, folic acid deficiency, vitamin B ₆ deficiency, vitamin Bl2 deficiency, intoxication by industrial toxins eg Carbon monoxide, mercury, lead, perchlorethylene, intoxications by drugs such as psychotropic drugs, anticholinergics, antihypertensives, anticonvulsants, beta-blockers, digoxin, immunovasculitis, exsiccosis, electrolyte imbalances - hyponatremia and hypernatremia -, rheologically caused cerebral circulatory disorders eg in polycythemia and multiple myeloma;
on (ii): chronic subdural hematoma, brain tumors, Parkinson's disease, Hydrocephalus aresorptivus, Huntington's disease, multiple sclerosis, chronic alcoholism, infectious diseases such as meningoencephalitis, lues, brain abscess, Creutzfeldt-Jakob disease or a dementia complex with Lewy bodies and
to (iii): depression, oligophrenia or schizophrenia.

By the disclosure of the present invention, the person skilled in the art can adult neurogenesis in connection with various diseases. determine. The classification of these diseases can be based on several Species take place. The person skilled in the art is known to have the abovementioned Classify diseases differently, e.g. in affective diseases, especially major depression and bipolar disorders, post-traumatic stress disorders, psychotic illnesses especially schizophrenia in their forms of expression; Autism, personality disorders, obsessive-compulsive disorder, hyperactivity Syndrome and attention disorders. But also prion diseases in their acquired or familial form are a dementia in the sense of the invention.

Important Dementia forms according to the invention are the primary cortical-degenerative Form of the Alzheimer's type and the innate (especially for example CADASIL) or acquired vascular forms dementia with concomitant symptoms: depression, sleep disorder, speech and movement disorders, Urinary and fecal incontinence as well as chronic constipation. It can be provided that the therapy of the invention in a holistic concept whose goal is the activation of the patient, the Improvement of his cognitive performance and compensation his disturbed social integration is.

Dementia within the meaning of the invention can be defined eg according to DSM IV; the person skilled in this classification of standard works is known in detail, it will therefore refer only to individual examples below:
Under 300 = organic and psychotic disorders, delusion and autism
over 300 = neuroses, and (almost) everything else

Interpretatio of the 2. Place (300-319):

• 0 = not organic, neurotic, reactive in genesis
• 1 = (suspected) mild organic causative factor

Interpretation of the 3. Job:

• 290 = Alzheimer's
• 291-2 = Substance deprivation or intoxication with delirium, delusion, organic disorders
• 293 = faults due to a medical disease factor
• 294 = other dementias
• 295 = schizophrenia and schizoaffective disorder
• 296 = affective disorders (not neurotic)
• 297 = delusion
• 298 = reactive and atypical psychoses
• 299 = Autism and other developmental disorders (unspecified)
• 300 = neuroses (in the classical sense)
• 301 = personality disorders
• 302 = sexual disorders (without medical illness factor)
• 303 = alcohol dependence and intoxication
• 304 = dependency of psychotropic substances
• 305 = substance abuse and intoxication
• 307 = special developmental and sleep disorders
• 308-9 = adjustment and stress disorders
• 310 = organic personality disorders
• 311 = depressive disorders (not closer designated)
• 312 = disturbances impulse control, monomania
• 314 = attention disorders and hyperactivity
• 315 = Speech, calculation and motor disorders in children
• 316 = physical conditions where mental factors play a role
• 317-9 = oligophrenia

Special coding guides (Selection):

• 332-3 = Neuroleptic side effects and consequences
• 347 = narcolepsy
• 607-25 = sexual dysfunction due to a medical disease factor
• 780 = sleep disorders due to a medical disease factor
• 780.09 = Delir (not closer designated)
• 780.9 = age-related cognitive decline
• 787 = Enkopresis with constipation and overflow incontinence

Especially preference is given to dementia development (cognitive decline) in the context of aging (decline in mental capacity, combined with fast fatigue, progressive disturbances of memory and orientation, etc.), especially dementia in the form from Alzheimer's. Alzheimer's disease in the sense of the invention is a multifactorial, looking for family or acquired senile, mostly in the 5th to 6th decade of life clinical manifesting form of dementia, pathologically characterized by a progressive cerebral cortex atrophy with senile plaques, degeneration of neurofibrillary tangles and congenital Angiopathy. Alzheimer's is in the context of the invention in certain circumstances with blurred heart symptoms (aphasia, apraxia) combined.

in the For the purposes of the invention, the term depression is defined very broadly. This term is not specific to age-related or endogenous or exogenous factors bound. Preference is given to major depression, not necessarily a recognizable physical illness as a cause must or with an external mental Reason reasonably is. Any form of sad mood, the feeling of Hopelessness and inner emptiness, the inhibition of will, thought and drive, if necessary also physically can be felt for example, as a weakness or heart anxiety, are endogenous depressions in the sense of the invention. Decreased concentration and attention as well as diminished Self-esteem and self-confidence, feelings of guilt and worthlessness as well as negative and pessimistic ones Future perspectives and suicidal thoughts, plans and / or actions, as well sleep disorders and reduced appetite are for the purposes of the invention symptoms, preferred Additional symptoms to depression diseases. Core symptoms are preferred a depressive mood in the sense that they are the average Professional is known, the loss of interest and pleasure as well the increased Fatigability. However, the invention is not limited to the characterization of Dementia or depression by determining the modification of the restricted to adult neurogenesis, all neurological or psychiatric disorders can be associated with a change associated with neurogenesis.

in the Meaning of the invention may be related to adult neurogenesis to be diagnosed or treated with the following conditions: neurodegenerative Diseases, in particular Parkinson's disease and other movement disorders, Alzheimer's disease and other dementias syndromes, tumors of the brain, especially glioblastoma multiforme, gangliogliomas and neurocytomas, addictions, especially alcohol, cannabis and opiate abuse, inflammatory diseases of the brain, especially multiple sclerosis, and the consequences of Reduced blood flow (ischemia, Stroke, high blood pressure) and brain injuries. The expert Other neurological disorders are known.

Prefers continues to be a more detailed analysis of temporal lobe epilepsy by the determination according to the invention adult neurogenesis. Temporal lobe epilepsy is a partial epilepsy with localization of neuronal discharge in the temporal or temporal lobe Share it. It shows focal and secondary or primary generalized motor symptoms or sensory type, with or without aura, especially psychomotor seizures, as they are known in the art.

at Epilepsy according to the invention may preferably be a the following epilepsies act to the adversive, akinetic, audiogene, audiotive, corticalis, cursiva, diffuse, diurna, emotional, focal, photogenic, generalized, genuine, hallucinatory, hereditary, idiopathic, laryngeal, limbic, major, menstrual, metabolic, minor, musicogenic, myoclonic, nocturnal, nonconvulsive, partialis, partialis continua, propulsives, psychogenic, psychomotor, salivatory, senile, sensory, symptomatic, tarda, traumatica, versive, visual, centrencephalic, cerebellar epilepsy.

In a further preferred embodiment In the invention, the nerve cells are granule cell neurons, in particular in the adult hippocampus of a mammal.

A preferred method is based z. For example, calretinin in those of interest To stain cells and to determine their number. This can be used as a direct measure of the number the newly formed nerve cells are taken. In the preferred Form is an immunohistochemical detection in the tissue section on the brain tissue of mice, the test substances, their effect on the regeneration of nerve cells to be examined, administered. The colored cuts are evaluated on a light microscope. The cells, the calretinin included are counted. This is done according to rules as they are known in the art and which allow an evaluation of samples. You get as a result the number of calretinin-positive cells per hippocampus. These correlates directly with the number of new nerve cells. An extra calretinin would be then synonymous with more nerve cell regeneration.

From this it turns out that the study is not immunohistochemical alone but also, for example, protein biochemical (Western Blot, HPLC) on homogenized samples of brain tissue.

Also The method may be modified to include calretinin not immunohistochemically visible in cells with antibodies to calretinin is made, but z. B. a transgenic animal model is used, in which the green fluorescent protein GFP or a similar reporter gene under regulatory Gene sections of Calretiningens is expressed. Calretinin-positive Cells would be thus directly visible in the tissue. You can turn microscopically analyzed and quantified or, for example, with Help of fluorescence activated cell sorters or magnetic separation extracted from the tissue and so on. For these preferred Variant of the method are appropriate regulatory sequences of calretining ("Calretinin promoter"), especially those the for the nerve cell-specific calretinin expression are responsible, identified and in a genetic construct (vector) so with connected to the reporter gene that when activated the regulatory Elements the information for the reporter gene is read and that corresponding to the reporter gene Reporter protein is formed by the cell. This protein is distributed in the cell and can be detected there. The reporter gene GFP or its related red fluorescent reporter genes, for example make the expressing cells more suitable when irradiated with light wavelength green (resp. red) light up. Thus, no antibody-mediated step is necessary to visualize the calretinin-expressing cells. To this to be able to use must with the help of the vector construct a transgenic animals, preferred a transgenic mouse, which are then examined can.

With appropriate radiological or nuclear medicine methods could be the Number of calretinin-positive cells also determined on living organism which make long-term studies possible over time would. In any case would correspond the proven value of calretinin expression is the extent of its elimination Neurogenesis. While the microscopic examination at the brain section at the same time qualitative and allows quantitative statements are quantitative with biochemical or imaging methods in particular Statements possible.

Of the Detection of calretinin can be detected in multiple labels with evidence other markers are combined so that statements about subgroups of calretinin-positive cells are possible. Especially preferred is the combination with MRP8, MRP14, Parvalbumin, 5100 and / or Calbindin. The markers MRP8, MRP14, parvalbumin and / or 5100 can also isolated for the detection of adult neurogenesis.

The The invention also relates to the use of transiently postmitotically expressed endogenous cell components for the determination of adult neurogenesis. Preference is given to adult neurogenesis in a full-grown Brain determined by calretinin expression.

The The invention also relates to a method for identifying a the adult neurogenesis modifying agent, the adult Neurogenesis according to the method of the invention carried out in the presence and / or absence of the agent and the modification of neurogenesis is detected and the modification is an indication of a neurogenesis modifying agent.

In a preferred embodiment of the invention this method further comprises the formulation the identified agent or derivative or homologue of which with a pharmaceutically acceptable carrier.

• a) in-Kontakt-bringen eines Mittel-Kandidaten mit Nervenzellen,
• b) Bestimmung der adulten Neurogenese,
• c) Abgleich der bestimmten Neurogenese mit einer Standard-Neurogenese und
• d) gegebenenfalls Vergleich, ob das Mittel die adulte Neurogenese modifiziert.

The invention also relates to an agent for modifying adult neurogenesis obtainable by

• a) contacting a middle candidate with nerve cells,
• b) determination of adult neurogenesis,
• c) matching the specific neurogenesis with a standard neurogenesis and
• (d) where appropriate, a comparison of whether the adult neurogenesis modified.

medium or pharmaceutical compositions used interchangeably herein can be are substances according to the invention and preparations of substances intended for use on or in the human or animal body, adult neurogenesis to modify. Medical adjuvants are according to the invention Substances used for production as active ingredients of medicines be used. Pharmaceutical auxiliaries serve the appropriate formulation of the drug or the pharmaceutical Composition and can even if only during needed in the manufacturing process, subsequently be removed or can be considered pharmaceutically acceptable carrier Be part of the pharmaceutical composition. Examples of pharmaceutical compatible carrier are known in the art.

The Drug formulation or formulation of the pharmaceutical Composition is optionally in combination with a pharmaceutically acceptable carrier and / or diluents. examples for suitable pharmaceutically acceptable carrier are known in the art and include e.g. Phosphate buffered Saline solutions, Water, emulsions, e.g. Oil / water emulsions, various types of detergents, sterile solutions, etc. Medicines or Pharmaceutical compositions comprising such carriers can by means of be formulated known conventional methods. These medicines or pharmaceutical compositions can be given to an individual in one be administered in a suitable dose, for example in one area of 1 μg up to 10 g of molecules per day and patient (human or animal). Preference is given to doses from 1 mg to 1 g. Preference is given to administering as few as possible and low doses, and more preferably a single dose, for example also a radioactively labeled molecule for diagnosis or examination a civilization disease.

The Administration can be done in several ways, for example intravenously, intraperitoneal, intrarectal, intragastrointestinal, intranodal, intramuscularly, locally, but also subcutaneously, intradermally or on the skin or over the Mucous membranes. The administration of nucleic acids - e.g. Antisense constructs - can also in the form of gene therapy done, for example, viral vectors. The type of dosage and the route of administration may be determined by the attending physician determined by the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, such as. the size, the Body surface, the Age, sex or general health of the patient, but also of the special remedy which is administered, the Duration and route of administration and other medications that possibly be administered in parallel.

The pharmaceutical compositions or the drug in particular, the agent found, its stimulators or agonists, Inhibitors or antagonists, recognition molecules against it, or / and these coding nucleic acid molecules in one suitable solution or administration form. these can either alone with the appropriate among medicines or used excipients described in pharmaceutical compositions become.

The pharmaceutical composition or the drug can of course also a Combination of two or more of the pharmaceutical according to the invention Compositions or drugs and a combination with other medicines timed in an appropriate manner administered or used together or separately. The preparation or pharmaceutical compositions of the medicaments carried out according to known methods.

Prefers it is to use the remedy as a diagnostic, prophylactic, therapeutic, post-treatment and / or leveling agent to use, for. B. to the treatment to follow the modification of adult neurogenesis.

The The invention also relates to a bioassay and a kit comprising recognition molecules which transiently post mitotic expressed endogenous cell constituents, such as calbindin, MRP8, MRP14 and / or parvalbumin, preferably calretinin, can be determined and detected. At the recognition molecules The kit or bioassay can be, for example, antibodies Antisense constructs, chelators, specifically interacting Lipids and carbohydrates or lectins, helix bundle proteins, Fusion proteins, and / or Lipocaline act, their interaction with the transiently postmitotically expressed endogenous cell constituents, prefers calretinin, so determinable that a statement about the adult neurogenesis possible is. Further preferred are MRP8, MRP14 or parvalbumin. Antibodies in Sense of the invention are in addition to complete antibodies single chain antibody fragments, Multibodies, Fab fragments, fusion proteins e.g. from an antibody fragment with Peptides or proteins and / or an immunoglobulin of the isotypes IgG, IgM, IgA, IgE, IgD and / or their subclasses. Here can it's murine, chimerized, Humanized, human, partially human antibody or antibody fragment act.

Of the Bioassay as well as the kit can include other chemical reagents that are used to detect the endogenous Cell components are required, it may preferably be e.g. to antibodies or fragments thereof which are immunological or histological Enable detection of adult neurogenesis. Within the meaning of the invention Bioassay is both a procedure and an arrangement or device comprising biocomponents or reagents for determination adult neurogenesis, understood; this double meaning of the term Assay is known in the art. The kit can combine information the contents of the kit and / or to provide a formulation of the kit Have kits. This information may be for example to handle a leaflet or to get information on one Homepage available is provided. Kit and assay can continuing excipients or carriers, Enzymes, marker substances but also storage containers, Slides, optical instruments, injection devices, microtomes and / or include other device parts that contain a biological, chemical and / or physical determination of adult neurogenesis or support.

in the Below, the invention will be explained in more detail with reference to an example, without being limited to this example to be.

Around to determine the time of calretinin expression, 24 six week old C57BL76 mice obtained from the Fa. Charles River and divided into 6 groups. The Animals received a single injection of bromodeoxyuridine (BrdU) and were 4 hours, 1 day, 3 days, 1 week, 2.5 weeks or 4 weeks later killed and removed the brain tissue.

Further 69 C57BL / 6 mice were divided into four groups: runner (RUN), delightfully alive (REIZ), with experimentally triggered seizures (ANF) and controls (KTR). RUN animals lived in twos or threes in cages normal size with unlimited Access to a wheel. Eight mice were kept in a charming environment, cf. previous publications (Van Praag et al., 1999). The day before BrdU injection (day 0) ANF animals received a systemic injection of kainic acid (KA, Fa. Sigma) at a dose of 30 mg / kg body weight in 0.1M phosphate buffer. While For the first seven days, all animals received a daily injection of 50mg / kg body weight BrdU in sterile physiological saline. All animals lived four Weeks after the last BrdU injection continue under the experimental Conditions of their group and were then examined.

The Animals were overdose Ketamine deeply anesthetized and perfused through the heart with a 4% paraformaldehyde solution. The brains were removed, stored in the fixan for a further 24 hours and then transferred to a 30% sucrose solution. Fourty Micron thick tissue scraps were from a dry ice cooled copper block with a Leica SM200R carriage microtome in a coronary orientation cut. For the BrdU immunohistochemistry, the sections were treated with 2N hydrochloric acid (HCl) for 30 Minutes at 37 ° C and then neutralized in 0.1M boric acid buffer (pH 8.5).

Around the number of cells labeled BrdU and expressing calretinin sections were cut with the peroxidase method (ABC system, Vectastain, Vector Laboratories) with biotinylated donkey anti-rat and anti-rabbit Antibodies and nickel-reinforced Diaminobenzidine as a dye. Triple labeling by immunofluorescence were after sooner Standard protocols described (Kempermann et al., 2003). When Primary antibodies were used: rat anti-BrdU (1: 500, Harlan Seralab), mouse anti-NeuN (1: 100; Chemicon), mouse anti-calretinin (1: 250; Swant), goat anti-calretinin (1: 250; Chemicon); rabbit anti-calretinin (1: 250; Swant), mouse anti-calbindin (1: 250; Swant), goat anti-doublecortin (1: 200; Santa Cruz Biotech), Goat anti-EAAT (1: 250; Chemicon), rabbit anti-Prox-1 (1: 5000; Gift of Dr. Samuel Pleasure, San Francisco), rabbit anti-Ki67 (1: 500, Novocastra).

When secondary Fluorescent antibody were used: anti-rat Rhodamine X, anti-mouse FITC, anti-mouse CY5, anti-rabbit CY5, anti-rabbit FITC, anti goat CY5 (all 1: 250; Jackson Laboratories, distributed by Dianova). Fluorescent sections were in polyvinyl alcohol with Diazobicyclo-octane raised as a bleach protection.

From all animals were given series with every sixth brain slice (240μm removed) for BrdU and calretinin stained with the peroxidase method. Positive cells were with a 40 × lens (Leica) in the rostro-caudal extent of the granule cell layer counted. As The classical optical dissecting method has been described so far modified that cells in the topmost level of focus as in focus appeared, not counted were (Kempermann et al., 2003). The obtained numbers were with 6 multiplied by the estimated total of the BrdU or calretinin positive cells per granule cell layer.

One-in-twelve series of brain slices from each animal in each group were tri-stained for BrdU, neuronal markers NeuN and calretinin (or other combinations, see above and below). Fluorescence signals were detected with a spectral confocal microscope (Leica TCS SP2). All analyzes were performed in sequential detection mode to avoid cross detection between the channels.

For each Animal and any combination of phenotypes 50 BrdU-labeled cells within the granule cell layer were co-expressed of the different markers. The relative numbers were on the absolute BrdU-positive Cells per grain cell layer relative to the absolute number of BrdU-labeled cells per phenotype receive. Accordingly was for Combinations of other markers except BrdU proceed.

All Numerical analyzes were performed on Statview 5.0.1 for the Macintosh. For all comparisons An analysis of variance (ANOVA) was performed followed by a Fisher post hoc test if allowed.

Results:

Four Weeks after BrdU injection expressed about 6% of BrdU-labeled Cells in the granule cell layer Calretinin. It is known that about 60 to 70% of the BrdU-labeled cells another calcium-binding protein, calbindin, at this time contained (Kempermann et al., 1997a). Calretinin-positive cells found themselves in the subgranular Zone of the granule cell layer. Morphologically they showed no noticeable differences to the adjacent calbindin-positive cells. At this time were all calretinin-positive cells also positive for neural markers NeuN. Also Calbindin positive cells were NeuN positive. So it was possible (based on the neuronal marker NeuN) two populations of newly formed, BrdU-labeled neurons identify: a group of calretinin-positive cells, and one Group of calbindinpositive cells.

If Calretinin expression at various times after BrdU injection examined wure, could be found that calretinin already found one day after BrdU injection in the BrdU-positive cells can be. The maximum was with over 75% of BrdU-positive cells reached about a week after the injection. Converted to absolute Values this means that, given the number of BrdU-positive cells first rises and then decreases again, the number of BrdU / calretinin double positive cells their peak reached three days after the injection. Six weeks after the injection no double-labeled cells could be found anymore. These Findings show that calretinin transient in BrdU-labeled cells is expressed.

doublecortin Another key marker is transient during adult hippocampal neurogenesis is expressed (Kempermann et al., 2003). On day 1 after BrdU injection was found a big one Percentage of BrdU-positive cells found at the same time as well calretinine and doublecortin were positive. Such an overlap did not exist after four weeks.

Of the Increase in the absolute number BrdU-positive calretinin-positive Cells over the first few days after the BrdU injection was parallel to an increase the BrdU / NeuN double positive Cells. While but after two and a half weeks they reached a stable number, decreased the number of BrdU / calretinin double positive cells again from. From the finding that after four weeks all calretinin-positive cells NewN expressed while too early Dates an overlap between calretinin and doublecortin, it can be concluded that Calretinin transient during earlier Developmental stages is expressed in newborn nerve cells.

granule cells are glutamatergic and therefore express the Excitatory Amino Acid Transporter (EEAT). They are also expressed by the expression of the transcription factor Prox-1 characterized (Pleasure et al., 2000). It showed be that already one week after BrdU all BrdU-positive calretinine expressing Cells positive for Prox-1 were and EAAT expressed. These findings prove that it is the calretinin-positive Cells around young granule cells is.

It Individual calretinin-positive cells were found that were also positive for calbindin were, and there were a few cells with this co-expression found. This speaks for a quick change between the two calcium-binding proteins while the maturation of the granule cells.

It were identifiable cells that were acutely in the cell cycle with an antibody against Ki67, a protein that during the entire cell division is expressed. We examined hundreds of calretinin-positive cells and did not find any Ki67 positive was. From this we conclude that calretinin expression is the time while the maturation of the new granule cells marked, in which the cells have left the cell cycle.

Finally, it was investigated whether stimuli known to induce adult hippocampal neurogenesis act in a similar manner on the easily determinable number of calretinin-positive cells. Voluntary physical activity (RUN), stimulus-rich environment (REIZ), experimentally induced seizures (ANF) and controls (KTR) were selected. REIZ has no or only a minor effect on cell division activity, but primarily promotes the survival of newborn cells (Kempermann et al., 1997b). RUN on the other hand robustly stimulates cell division activity (Van Praag et al., 1999). Kainic acid-induced seizures, such as in ANF, are among the strongest stimulators of cell division activity in the dentate gyrus (Parent et al., 1997).

In In all three paradigms, the net neurogenesis was four weeks after BrdU increased and this increase was reflected in the absolute number of calretinin-positive cells within the granule cell layer again. REIZ showed no significant (but descriptively recognizable) Rise, with the primary survival Effect is compatible. In ANF, the increase was in CR-positive cells about fifteenfold. RUN ranked between these extremes.

Taken together, show these findings that the number of calretinine expressing Cells in the dentate gyrus of the mouse as a surrogate marker for the expiring Adult neurogenesis can be taken as calretinin transient is expressed in adult-born granule cells, from the time the cells leave the cell cycle have until about two to four weeks later.

References

• Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., and Gage, F.H. (2003). Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development 130, 391-399.
• Kempermann, G., Kuhn, H.G., and Gage, F.H. (1997a). Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci USA 94, 10409-10414. Kempermann, G., Kuhn, H.G., and Gage, F.H. (1997b). More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493-495.
• Parent, J.M., Yu, T.W., Leibowitz, R.T., Geschwind, D.H., Sloviter, R.S., and Lowenstein, D.H. (1997). Dentate granule cell neurogenesis is increased by seizures and de facto aberrant network reorganization in the adult rat hippocampus. J Neurosci 17, 3727-3738.
• Pleasure, S.J., Collins, A.E., and Lowenstein, D.H. (2000). Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development. J Neurosci 20, 6095-6105.
• Van Praag, H., Kempermann, G., and Gage, F.H. (1999). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2, 26.6-270.

Claims (20)

Method for the determination of an adult neurogenesis, characterized in that in at least one nerve cell transiently postmitotically expressed endogenous cell constituents are determined. Method according to claim 1, characterized in that that the cell constituents are selected are from the group comprising calbindin, MRP8, MRP14, parvalbumin, 5100 and / or calretinin. Method according to claim 1 or 2, characterized that the cell constituent is calretinin. Method according to one of the preceding claims, characterized characterized in that the nerve cells are selected from the group comprising adendritic nerve cells, apolar nerve cells, bipolar neurons, multipolar nerve cells, polyneuritic nerve cells, pseudounipolar neurons, unipolar nerve cells, neurosecretory nerve cells, preganglionic nerve cells, postganglionic nerve cells, amacrine cells, horizontal cells, bipolar cells, Golgi cells, Purkinje cells, Motor neurons, hair, pyramid and / or basket cells, in particular granule cells and / or interneurons. Method according to the preceding claim, characterized characterized in that the nerve cells neurons, in particular granule cells, of the hippocampus. Method according to one of the preceding claims, characterized characterized in that the cell components are invasive and / or non-invasive be determined. Method according to the preceding claim, characterized characterized in that the invasive determination is a histological and / or an immunohistochemical determination and the non-invasive Determination of a magnetic resonance tomography, computed tomography, SPELT and / or positron emission tomography. Method according to one of the preceding claims, characterized characterized in that it is in the absence and / or presence of funds carried out whose influence on adult neurogenesis will be tested should. A method of identifying an adult neurogenesis modifying agent, characterized in that adult neurogenesis according to the method of any of claims 1-8 is carried out in the presence and / or absence of the agent and the modification of the neurogenesis is detected and the modification is an indication of a neurogenesis modifying agent. Method according to the preceding claim, characterized characterized in that it continues to be the formulation of the identifying By means of or a derivative or a homologue thereof with a pharmaceutically acceptable carrier includes. Means that modifies adult neurogenesis, available by a) contacting a middle candidate with nerve cells, b) Determination of adult neurogenesis according to the method of the claims 1-8 c) matching the specific adult neurogenesis with a Standard neurogenesis and d) if applicable, compare whether the Means modified adult neurogenesis. Use of transiently postmitotic expressed endogenous cell components for qualitative and / or quantitative Determination of neoplasm of nerve cells in the peripheral, central and / or autonomic nervous system, especially in the hippocampus and / or olfactory bulb. Use according to the preceding claim, characterized characterized in that the neoplasm of nerve cells in an adult Brain is determined after birth. Use according to claim 12 or 13, characterized that the transient postmitotic expressed endogenous cell constituent MRP8, MRP14, parvalbumin, 5100 and / or calretinin. Use according to any one of claims 12-14, characterized in that for the detection of calbindin, MRP8, MRP14, parvalbumin, S100 and / or preferred calretinin is a transgenic animal model used in the regulatory sections of calbindin, MRP8, MRP14, S100 and / or calretining control the expression of a reporter gene. Use according to any one of claims 12-15, characterized that the detection is calbindin, MRP8, MRP14, parvalbumin, S100 and / or prefers calretinin-positive Cells by isolation of the cells via the expression of the reporter gene preferably in fluorescence activated cell sorter (FACS) and / or with Magnet separation takes place. Bioassay for the determination of adult neurogenesis, characterized in that it comprises recognition molecules linked to calretinin Calbindin, MRP8, MRP14, S100 and / or parvalbumin specific and detectably interact. Bioassay according to claim 17, characterized that the recognition molecules Antibody, Lectins, chelators and / or anti-sense constructs are. A kit comprising a bioassay according to claim 17 or 18 optionally with information for combining the contents of the kit. Use of the kit according to claim 19 for the determination adult neurogenesis.