DE102009054057A1 - Screening method for active ingredients for the prophylaxis and therapy of neurodegenerative diseases - Google Patents

Abstract

A screening method for active ingredients for the prophylaxis and therapy of neurodegenerative diseases, preferably Alzheimer's disease, is described, the active ingredients inhibiting the transport of Aβ42 into the cell, preferably the cell nucleus. Diagnostic and therapeutic applications based thereon are also described.

Description

The present invention relates to a screening method for drugs for the prophylaxis and therapy of neurodegenerative diseases, preferably Alzheimer's disease, wherein the drugs inhibit the transport of Aβ42 into the cell, preferably the nucleus.

Alzheimer's disease (AD) is a neurodegenerative disease and is the most common form of dementia. The most important pathological feature of Alzheimer's disease is amyloid plaques. The amyloid plaques consist of extracellular aggregates of the amyloid β-peptide (Aβ), which is a cleavage product of the amyloid precursor protein (APP). Soluble Aβ peptides are a specific feature of AD. The soluble forms can oligomerize and are then detectable as dimers, tetramers, and up to dodecamers and higher aggregates, and can then further aggregate into fibrils. These fibrils are found z. In the amyloid plaques in the brain of patients.

The research shows that the soluble forms of Aβ with a length of 42 residues (Aβ42), and in particular the Aβ42 dimers, trimers and tetramers, are responsible for neuronal loss and therefore cognitive performance loss, but not fibrils or plaques , A relationship to the pathological features (plaques and neurofibrillary tangles) at the molecular level could not be directly demonstrated.

Despite numerous in vitro studies, the molecular mechanism of toxicity has remained undetected. The toxicity of the oligomeric forms is measured as cell death and inhibition of long term potentiation (LTP) in cell culture systems and animal models. The resulting hypotheses suggest that (a) Aβ oligomers form so-called pores via hydrophobic interaction with lipids in cell membranes, which destroy the membranes and unbalance the ion balance, or (b) cell death via a receptor-mediated mechanism cause ( Walsh et al. (2007) J. Neurochem. 101, pp. 1172-1184 )

Munter et al. ( Munter et al. (2007), EMBO J. 26, pp. 1702-1712 ) showed that APP contains three consecutive GxxxG (glycine, three arbitrary amino acids except proline, glycine) dimerization motifs, with the motifs at Aβ positions G29 and G33 playing an essential role in the processing of the APP. It could be shown that by point mutation of the glycine to an alanine no longer predominantly Aβ42 is formed, but there is a sequential further cutting of the peptide, resulting in shorter Aβ species such as Aβ1-38 and Aβ1-34. Furthermore, it could be shown that this toxic effect can be reversed if the glycine at Aβ position 33 is mutated to an alanine or isoleucine. These findings provide good approaches for the development of strategies for the prophylaxis or therapy of Alzheimer's disease. However, due to the urgency of the possibility of efficient treatment / prophylaxis of Alzheimer's disease, it is important to look for other therapeutic concepts.

Thus, the technical problem underlying the present invention is to provide methods of identifying active substances which are suitable for the prophylaxis or therapy of Alzheimer's disease or other neurogenic diseases.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

It has been found by the inventors that Aβ peptides can be taken up in the cell and exert their toxic effect there. In the investigations leading to the present invention it could be shown that both Aβ42 and Aβ42G33A are not only taken up by the cells but are transported directly into the cell nucleus. The experimental data show that the toxic peptide Aβ42 accumulates in the nucleus of human neuroblastoma cells and can exert a transcriptional regulatory function. In the investigated examples, it could be shown that Aβ42 acts specifically suppressing the transcription of LRP1 and KAI1 but activating the transcription of APP. It could further be shown that the non-toxic peptide Aβ42G33A has no influence on the expression of the mRNA levels of the examined genes. From the familial cases of AD studied it is known that an increased production of Aβ42 and a changed ratio of the Aβ species Aβ40 and Aβ42 may be responsible for AD. It is unclear why Aβ42 plays a key role in neuronal loss and results in cognitive deficits. It is believed that Aβ42 may also influence genes other than those shown, which play a crucial role in learning and memory, and thus serve as therapeutic targets. Due to the new finding that Aβ42 takes on a regulatory function in the nucleus, new approaches to the diagnosis and therapy of Alzheimer's disease are possible.

• (1) Screening nach Genen, die für das Lernen und das Gedächtnis unerlässlich sind und die durch Aβ42 oder andere in den Kern importierte Aβ-Formen reguliert werden.
• (2) Screening nach zytoplasmatischen Transportmolekülen, die den späteren Kernimport von Aβ42 oder andere in den Kern importierte Aβ-Formen ermöglichen.
• (3) Identifizierung von Inhibitoren für den Kernimport von Aβ42, um die Gen-regulatorische Wirkung von Aβ42 oder andere in den Kern importierter Aβ-Formen zu verhindern.

The findings obtained in the present invention find, inter alia, the following applications:

• (1) Screening for genes that are essential for learning and memory and that are regulated by Aβ42 or other Aβ forms imported into the nucleus.
• (2) Screening for cytoplasmic transport molecules that facilitate later nuclear import of Aβ42 or other Aβ forms imported into the nucleus.
• (3) Identification of inhibitors for the nuclear import of Aβ42 to prevent the gene regulatory effect of Aβ42 or other Aβ forms imported into the nucleus.

• (4) Aktivierung des Ubiquitin-Proteasom-Systems, das am Abbau von amyloiden Aggregaten von Aβ42 analog zu pQ Aggregaten im Kern beteiligt ist.

Recently, the autosomal dominant inherited polyglutamine disorder, in which polyglutamine aggregates form in the nucleus, has been shown to be much more toxic in the cell nucleus than aggregates located in the cytosol ( Iwata et al. (2009), Journal Biol. Chem. 284, pp. 9796-9803 ). The degradation of aggregates in the nucleus involves ubiquitin ligases, such as San1p and UHRF-2, which reverse poly-Q-induced toxicity. Analogously, the following results for the present invention:

• (4) Activation of the ubiquitin-proteasome system involved in the degradation of amyloid aggregates of Aβ42 analogous to pQ aggregates in the nucleus.

• (a) Diagnose (Nachweis der Akkumulation von Aβ42 im Zellkern (z. B. über Biopsien)); und
• (b) Therapie (Verhinderung der Aufnahme von Aβ42 (oder anderen Gen-regulatorischen Formen von Aβ) in die Zelle, bzw. den Kern, Verhinderung bzw. gezielte Beeinflussung der Gen-regulatorischen Wirkungen von Aβ42 (oder anderen Gen-regulatorischen Formen von Aβ), Aktivierung von Abbaumechanismen im Zellkern, die auf Aβ42 wirksam sind).

The present findings are also useful for:

• (a) diagnosis (detection of accumulation of Aβ42 in the cell nucleus (eg via biopsies)); and
• (b) Therapy (to prevent the uptake of Aβ42 (or other gene regulatory forms of Aβ) into the cell, or the nucleus, to prevent or specifically affect the gene regulatory effects of Aβ42 (or other gene regulatory forms of Aβ ), Activation of nuclear degradation mechanisms that are active on Aβ42).

Brief description of the figures

Fig. 1: Time-dependent accumulation of A) Aβ42 and B) Aβ42G33A in the nucleus of human neuroblastoma cells

The quantification was carried out by ELISA as a relative amount to the value at 0 hours assumed to be 1.0, d. H. without addition of synthetic peptide.

Fig. 2: mRNA expression after incubation with Aβ42

Using RT-PCR, cDNA was generated from isolated mRNA. mRNA expression of LRP1 and KAI1 showed a significant decrease after 4 and 6 h incubation of the cells with Aβ42. After 8 h of incubation, there was a significant increase in APP. The mRNA levels of BACE1 and Hes1 were not affected.

Fig. 3: mRNA expression after incubation with Aβ42G33A

Using RT-PCR, cDNA was generated from isolated mRNA. Aβ42G33A has no effect on mRNA expression of LRP1, KAI1, Hes1, APP and BACE1.

Fig. 4: Chromatin immunoprecipitation (ChIP) of DNA fragments of the promoters of LRP, KAI1 and Hes1

• Input: used DNA. Controls: αIgG, αp50.
• (A) Hes1 promoter: no interaction of Aβ and the promoter could be detected at any time.
• (B) LRP1 promoter: after 0.5 h incubation with Aβ42, Aβ42 could be detected on the promoter. After 1 h Tip60 and Fe65 were detected. After 8 h incubation with Aβ42 Tip60 and Fe65 could no longer be detected on the promoter, Aβ42 remains on the promoter.
• (C) KAI1 promoter: after 1 h of incubation with Aβ42, this was detected on the promoter. The co-repressor N-CoR was no longer detected. After 2 h of incubation, Tip60 and Fe65 were detected. After 6 h of incubation Tip60 and Fe65 could no longer be detected on the promoter, the co-repressor N-CoR was not detected, Aβ42 remains on the promoter.

• (a) Inkontaktbringen von Zellen in Gegenwart von (i) Aβ42 und (ii) eines Wirkstoffkandidaten unter Bedingungen, die deren Aufnahme durch die Zellen erlauben; und
• (b) Bestimmung der zellulären oder subzellulären Lokalisation von Aβ42 in den Zellen;

wobei eine verringerte zelluläre oder subzelluläre Lokalisation von Aβ42 im Vergleich zu einer Kontrolle ohne Wirkstoffkandidaten darauf hinweist, dass der Wirkstoffkandidat für die Prophylaxe/Therapie einer neurodegenerativen Erkrankung geeignet ist.Thus, the present invention relates to a method for the screening of active substances for the prophylaxis and / or therapy of a neurodegenerative disease, the method comprising the following steps:

• (a) contacting cells in the presence of (i) Aβ42 and (ii) an agent candidate under conditions permitting their uptake by the cells; and
• (b) determining the cellular or subcellular localization of Aβ42 in the cells;

wherein a decreased cellular or subcellular localization of Aβ42 in comparison to a control without drug candidates indicates that the drug candidate is suitable for the prophylaxis / therapy of a neurodegenerative disease.

The optimal times between steps (a) and (b) can be determined by a person skilled in the art by routine experiments and are generally between 20 minutes and several hours (uptake into the cell) or detection by cell fractionation (hours to one day , Centrifugation and then ELISA or Western blot, about 1 day). These are work processes that are standardized in the laboratory and not just apply to the experiments described here.

In the screening method of the present invention, Aβ42 and the drug candidate may be administered simultaneously or sequentially (with a time delay), e.g. B. the drug candidate before Aβ42.

The term "neurodegenerative disease" as used herein includes any form of neurodegenerative diseases or dementias such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, Lewy body dementia, Pick's disease (frontotemporal dementia), progressive supranuclear palsy, corticobasal degeneration, and Multiple sclerosis (MS).

The cells used in the method of the invention can be derived from a variety of sources, e.g. For example, mammals (e.g., rodents, humans, primates, etc.) are chickens, etc. Preferably, the cells used in the method of the invention are mammalian cells, with human cells being preferred.

Especially preferred are neuronal cells, with neuroblastoma cells being most preferred. An example of a particularly preferred neuroblastoma cell is an SH-SY5Y cell. Other suitable cell lines are all common in the laboratory cell lines, z. CHO, HEK293, COS7, fibroblasts or N2a.

The cells used in the screening method according to the invention may be part of a non-human organism (animal model), a tissue or in cell culture. Non-neuronal cells may be primary cell cultures or cell lines derived therefrom. The non-neuronal cells and the neuronal cells may be autologous, allogeneic or xenogeneic.

The cells can be cultured in well-known growth media. For example, mammalian cells may be maintained in an in vitro culture in various nutrient media suitable for mammalian cells, for example neurobasal or L15 media (Life Technologies, Rockville, Maryland), which may be supplemented by various compounds, e.g. Antibiotics, vitamins, trophic factors, fetal serum, etc. Preferably, the cells are cultured in neurobasal medium supplemented with nutrients and growth factors. The cells can be kept for several days or weeks without losing their properties. Preferably, they are used shortly after their preparation, d. H. preferably within one week of preparation.

In general, the cells are exposed to the candidate compound (s) between 1 nM and 1 mM, for Aβ42 concentration in the range nano- to micromolar concentrations, which is below the LD concentration for each cell line (depending on the particular laboratory z B. at SY5Y cells at about 10 μM). Furthermore, each compound can be tested in parallel at different concentrations. Also, if necessary, various additives and / or vectors and / or products that help the compounds to enter the cells may be added, e.g. Liposomes, cationic lipids or polymers, penetratin, Tat PDT, peptides from adenoviruses (eg penton) or other viruses, etc.

The contacting can be carried out in known carriers, for. As plates, tubes, pistons and the like. In general, the contacting takes place in plates with numerous holes, which make it possible to perform numerous experiments in parallel. Typical carriers are. z. Microtiter plates, especially the 384-well microtiter plate format, which is easy to handle.

As used herein, the term "contacting cells" refers to contacting cells that act as an organism, i. H. as a non-human organism, preferably mammalian or, for example, as a cell culture maintained under conditions permitting uptake of the drug candidate (and Aβ42) into the cells.

As used herein, "in the presence of Aβ42 under conditions permitting its uptake by the cells" also includes the uptake of Aβ42 into the cell nucleus, where (a) Aβ42 can be externally supplied, for example via the culture medium, or (b ) is present in the cytoplasm after expression of a heterologous nucleic acid encoding Aβ42 with which the cell was previously transformed.

The determination of the cellular or subcellular localization of Aβ42 in the cells may be by routine methods, e.g. B. by specific binding to Aβ42 ligands, preferably antibodies, after previous cell fractionation, z. B by ELISA, Western blot or by microscopic techniques via fluorescent labeling together with the corresponding markers such as nuclear-specific proteins or DNA, and other methods described in more detail below. With regard to the above-mentioned methods and the following methods, all of which are routine methods, reference is made to standard works of the Referenced laboratory technology, z. B. "Molecular Biomethods Handbook", Edit .: J. Walker, Ralph Rapley, Human Press, 2nd. Edition, 2008 , or Janeway "Immunology", Spektrum Verlag, 7th edition, 2009 ,

The term "drug candidate" as used herein refers to previously known or unknown substances, wherein the substances to be investigated are in no way limited, i. that is, they can be very different test compounds. These may be naturally occurring and synthetic, organic or inorganic compounds as well as polymers (eg, oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides, and nucleotide analogs, analogous naturally occurring structures (eg, peptide imitators, nucleic acid analogues, etc.) and other compounds.

As starting material for possibly useful test compounds also extracts of natural products can be used. Such extracts may come from different sources, e.g. Fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. Activity-containing extracts can then be further analyzed for isolation of the active ingredient.

The test compound may be a single compound or a mixture. Thus, the test compounds can also be screened on a large scale. Thus, in a preferred embodiment of the screening method of the invention, the test compounds are part of a substance library. Depending on the configuration of the method, the test compounds may be added to a culture medium, injected into a cell, etc.

If a sample has been identified with a useful test compound or mixture in the screening method of the present invention, either the test compound in question can be isolated therefrom, or the original sample, if containing a mixture of different compounds, can be fractionated to give the number of different compounds per molecule Reduce the sample. The process according to the invention is then repeated with the individual fractions.

Assay formats for identifying positive test compounds are well known and may be modified as necessary by the skilled artisan, if necessary.

The compounds isolated by means of the screening method according to the invention are suitable as therapeutic agents for the treatment, prevention, control or alleviation of a neurological disease, preferably AD, or these can serve as so-called "lead structures" for the development of analogous compounds, i. h., agents having additional desired properties, for. B. the blood / brain barrier can pass well, or lose certain undesirable side effects or have a higher half-life (clearing rate). In addition, such optimized compounds may have a stabilized electron configuration and molecular conformation wherein the key functional groups may be presented substantially as in the parent compound. Preferably, the analogous compounds have spatial electronic properties comparable to the binding region of the parent compound but are smaller, e.g. B. only a molecular weight below 2 kD, preferably below 1 kD. The preparation / identification of such analogous compounds can be carried out by conventional methods, for. B. SCF analysis or CI analysis. The pharmacological properties of these analogous compounds can also be determined by known methods. Such agents may, for. B. the import of Aβ42 in the cell, preferably inhibit the nucleus, so as. B. to prevent the gene regulatory effect of Aβ42 or other Aβ forms imported into the nucleus.

In a further preferred embodiment of the method according to the invention, the localization of Aβ42 in the cell nucleus is determined. This can be done by conventional detection methods, for. B. ELISA, Western blot, fluorescence cytometry. Preferably, the determination of the localization after step (b) before the cells were fractionated subcellularly and the individual cell fractions, z. For example, the cell nuclei containing fraction can be tested separately for the presence of Aβ42.

In a particularly preferred embodiment of the method according to the invention, the detection of Aβ42 is based on the binding of a specific ligand, which is preferably provided with a detectable marker. Most preferred is an immunological method. Thus, the presence / concentration of Aβ42 can be determined by contacting the sample with an anti-Aβ42 antibody or a fragment thereof and then determining the binding of the anti-Aβ42 antibody or fragment thereof to Aβ42. The suitable antibodies may be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this context, the term "fragment" means all parts of the monoclonal antibody (eg Fab, Fv or "single chain Fv" fragments) which have the same epitopic specificity as the complete antibody. The preparation of such fragments is known to the person skilled in the art. Preferably, the antibodies are um monoclonal antibodies. The antibodies may be prepared according to standard methods, preferably using Aβ42 or a synthetic fragment thereof as immunogen. This polypeptide or peptide or the fragment thereof may, for. B. by obtaining the corresponding gene, cloning and recombinant expression can be generated. Methods for obtaining monoclonal antibodies are known in the art.

In an even more preferred embodiment of the method according to the invention, the Aβ42 concentration is determined via a primary monoclonal anti-Aβ42 antibody and a commercially available secondary biotin-conjugated antibody and streptavidin-peroxidase.

In a further preferred embodiment of the method according to the invention, the cell sample is immobilized. The cell sample may also be absorbed to the wall of a plastic dish by conventional methods such that Aβ42 does not lose its binding specificity, such as for a specific antibody.

In a further preferred, alternative embodiment of the method according to the invention, the anti-Aβ42 antibody or the fragment thereof are immobilized, i. h. that he z. B. is absorbed on the wall of a plastic shell without losing its binding specificity for Aβ42.

The detection of the binding of the antibody or the fragment thereof can be carried out by conventional methods, for. Western blotting, ELISA, radioimmunoassay (RIA) etc., with RIA and ELISA being preferred.

In a further preferred embodiment of the method according to the invention, the (reduced) localization of Aβ42 in the nucleus is indirectly determined by its gene regulatory effects, wherein preferably the gene regulatory activity (a) is an inhibition of the expression of LRP1 and / or KAI1, and / or (b) is an increase in the expression of APP. The change in expression can be determined by conventional methods, for example at mRNA level. The RNA concentration can be measured using specific probes / primers using Northern blot, RT-PCR, etc. On the other hand, the change in expression can also be measured at the protein level using detection methods such as those described above for the detection of Aβ42.

Finally, the present invention also relates to the use of a ligand specific for Aβ42, preferably an antibody as described above or a fragment thereof for the production of a diagnostic agent for determining the presence of a neurodegenerative disease or for determining the risk of developing a neurodegenerative disease. The concentration of Aβ42 in a patient cell, preferably in the cell nucleus, is determined and compared with a control derived from a healthy patient, preferably a collective of healthy patients from whom a mean value was determined. The sample can be taken from the patient by conventional methods, for example via a biopsy or from cerebrospinal fluid and / or blood.

Since it has been shown in the experiments leading to the present invention that Aβ42 is believed to exert its deleterious effects by (a) reducing gene expression of LRP1 and / or KAI1 and / or (b) increasing APP gene expression the present invention also relates to the use of a gene expression of LRP1 and / or KAI1 increasing and / or the gene expression of APP inhibitory compound for the manufacture of a medicament for the prophylaxis and / or therapy of a neurodegenerative disease. Suitable agents for modulating these genes, the sequence of which is known, can be identified by those skilled in the art by routine methods. The inhibition of gene expression can be carried out, for example, via siRNA, antisense RNA, ribozymes, etc. On the other hand, the gene products can be inactivated, z. B. on neutralizing antibodies.

For a therapy, gene expression modulating agents are preferably present in combination with a pharmaceutically acceptable carrier. Suitable carriers include, for example, phosphate buffered saline, water, emulsions, for example, oil / water emulsions, wetting agents, sterile solutions, etc. Administration may be oral or, preferably, parenteral. Methods for parenteral administration include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The appropriate dosage will be determined by the attending physician and will depend on various factors, such as age, gender, weight of the patient, stage of the neurogenic disease, mode of administration, etc.

The following examples illustrate the invention.

example 1

Both the toxic Aβ42 and the non-toxic Aβ42G33A are accumulated in the nucleus of human neuroblastoma cells.

Based on the hypothesis that Aβ peptides can be taken up into the cell and exert their toxic effect there, it could be shown that both Aβ42 and Aβ42G33A are not only taken up by the cells but are transported directly into the cell nucleus. Furthermore, shorter Aβ species such as Aβ38 and Aβ40 but also the longer form Aβ43 are transported into the cell nucleus. It is an active transport process that can be inhibited at 4 ° C. In addition, it has been shown that both the toxic Aβ42 and the non-toxic Aβ42G33A form accumulate in the nucleus of human neuroblastoma cells. Aβ42 and Aβ42G33A were detected in the cell nucleus by means of ELISA (enzyme-linked immunosorbent assay) ( 1 ). The graph shows that both peptides accumulate in the nucleus of SH-SY5Y cells in a time-dependent manner.

Example 2

Aβ42 exerts a transcriptional regulatory function in the nucleus

Based on the new finding that both Aβ42 and Aβ42G33A are to be detected in the nucleus and accumulate there in a time-dependent manner, it was investigated whether Aβ42 and Aβ42G33A have a gene-regulatory effect. Therefore, mRNA was isolated from SH-SY5Y cells and generated by RT-PCR (reverse transcription polymerase chain reaction) cDNA from this mRNA and amplified with the real-time PCR method in several cycles. In this particular method of PCR, a fluorophore is used whose intensity is proportional to the amount of reaction product produced so as to measure and determine the amplificate directly during the reaction. It could be shown that the incubation of the cells with the toxic peptide Aβ42 led to a significant decrease in the mRNA of two specific genes, LRP1 and KAI1, and that there was a significant increase in the mRNA of the APP. In addition, it could be shown that Aβ42 has no influence on the mRNA expression level of BACE1. As a control, the expression level of the mRNA of Hes1 was measured, which was not influenced by Aβ42 ( 2 ).

Studies of the mRNA expression levels of Neurobastoma cells treated with the non-toxic peptide Aβ42G33A revealed that there was no variance in mRNA expression ( 3 ). In addition, it could also be shown that both the shorter Aβ species, such as Aβ38 and Aβ40, as well as the longer Aβ43, also had no influence on the mRNA expression of the examined genes.

This result leads to the conclusion that Aβ42 can exert a transcriptional regulatory function in the cell nucleus, as demonstrated by the examples LRP1, KAI1 and APP.

Chromatin immunoprecipitation experiments (ChIP) showed that Aβ42 binds to certain regions of the DNA, promoters. It comes to the recruitment of the histone acetyltransferase Tip60 and the adapter protein Fe65 and probably to the complex formation with these proteins and the DNA ( 4 ). The interaction of Aβ42 with the promoters of LRP1 and KAI1 was identified as a specific event since no interaction with Aβ42 could be detected at the Hes1 promoter.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Walsh et al. (2007) J. Neurochem. 101, pp. 1172-1184 [0004]
• Munter et al. (2007), EMBO J. 26, pp. 1702-1712 [0005]
• Iwata et al. (2009), Journal Biol. Chem. 284, pp. 9796-9803 [0010]
• "Molecular Biomethods Handbook", Edit .: J. Walker, Ralph Rapley, Human Press, 2nd. Edition, 2008 [0027]
• Janeway "Immunology", Spektrum Verlag, 7th Edition, 2009 [0027]

Claims (15)

A method for the screening of drugs for the prophylaxis and / or therapy of a neurodegenerative disease, said method comprising the steps of: (a) contacting cells in the presence of (i) Aβ42 and (ii) an agent candidate under conditions permitting their uptake by the cells; and (b) determining the cellular or subcellular localization of Aβ42 in the cells; wherein a decreased cellular or subcellular localization of Aβ42 in comparison to a control without drug candidates indicates that the drug candidate is suitable for the prophylaxis / therapy of a neurodegenerative disease. The method of claim 1, wherein the cells are neuronal cells. The method of claim 2, wherein the cells are neuroblastoma cells. The method of claim 3, wherein the neuroblastoma cells are SH-SY5Y cells. Method according to one of claims 1 to 4, wherein the localization of Aβ42 is determined in the cell nucleus. Method according to one of claims 1 to 4, wherein prior to step (b) the cells are fractionated subcellularly and the detection of Aβ42 takes place in the nuclear fraction. The method of claim 6, wherein the detection is immunological detection. The method of any one of claims 5 to 7, wherein the decreased localization of Aβ42 in the nucleus is determined indirectly by its gene regulatory effects. The method of claim 8, wherein the gene regulatory effect (a) is an inhibition of the expression of LRP1 and / or KAI1, and / or (b) an increase in the expression of APP. Use of a specific Aβ42-binding ligand for the preparation of a diagnostic agent for the determination of a neurodegenerative disease or for determining the risk of developing a neurodegenerative disease. Use according to claim 10, wherein the ligand is an antibody. A ligand according to claim 10 or 11 for use in a method of diagnosing a neurodegenerative disease. Use of a gene expression of LRP1 and / or KAI1 increasing and / or the gene expression of APP inhibitory compound for the manufacture of a medicament for the prophylaxis and / or therapy of a neurodegenerative disease. Gene expression of LRP1 and / or KAI1-enhancing and / or APP inhibitory gene expression for use in a method of preventing and / or treating a neurodegenerative disease. A method or use according to any one of the preceding claims, wherein the neurodegenerative disease is Alzheimer's disease.

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