AT410673B - Detecting Alzheimer's disease and Down's syndrome, from underexpression of phosphoprotein ARPP-19, in tissue or body fluid - Google Patents

Abstract

Detecting Alzheimer's disease or Down's syndrome comprises determining, in a tissue or body fluid sample, from a human at risk, the content of the cyclic adenosine monophosphate (cAMP)-regulated phosphoprotein of molecular weight 19 kD (ARPP-19) and comparing this with a reference level. An Independent claim is also included for a kit for detecting ARPP-19 comprising a molecule that detects ARPP-19 and a reference value.

Description

AT 410 673 B

The invention relates to methods for the detection of neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimer's disease is an increasingly important public health problem. Alzheimer's disease alone is thought to have around 12 million people worldwide with the disease, with the number of these diseases increasing as life expectancy increases. It is estimated that between 40 and 50 million illnesses worldwide are expected by 2050. However, neurodegenerative dementias such as Alzheimer's are not always easy to diagnose. There are dozens of options that can be associated with dementia, such as Depression, drug use, dehydration, anemia, syphillis, viral infections and vitamin deficiency. Reliable diagnoses - the basic prerequisite for successful therapy - are therefore often difficult to make. Psychological test methods are currently used primarily to diagnose dementias such as Alzheimer's. In the post-mortem diagnosis of neurodegenerative diseases such as Alzheimer's, morphological methods are the focus.

Accordingly, it is an object of the present invention to provide simple and reliable diagnostic methods for neurodegenerative diseases which, alone or in combination with previously used methods, can be used for more precise, faster and more reliable diagnostics, in particular for more efficient and precise differential diagnostics.

This object is achieved according to the invention by a method for detecting Alzheimer's disease or Down syndrome, which is characterized by the following steps: - Providing a tissue sample or a body fluid of a person with suspected Alzheimer's disease or Down syndrome. - Determining the content of the cAMP-regulated phosphoprotein with a molecular weight of 19,000 (ARPP-19) in the tissue sample or in the body fluid and - Comparing the determined ARPP-19 content with a reference value with which the determination of the presence of Alzheimer's Disease or Down syndrome is possible in this person.

A whole range of substrates of the cAMP-dependent protein kinase (PKA) are known, which are also found in a wide variety of brain regions. However, little is known about the physiological or pathological function of these substrates.

In the context of the present invention, it has now been possible to demonstrate that a specific substrate for PKA, namely ARPP-19, is significantly downregulated in patients with Alzheimer's disease or Down syndrome. It has been shown that lower levels of ARPP-19 and decreased PKA activity are involved in the pathomechanisms of neurodegenerative diseases, and at a relatively early stage. It was shown that this disturbance of the cAMP-related signal transmission and phosphorylation result in a good correlation with the neurodegenerative diseases. This is all the more surprising considering that other PKA substrates, e.g. ARPP-16 (Horiuchi et al., J. Biol. Chem. 265 (16), 9476-9484) shows no such marker function as ARPP-19.

According to the invention, it was found that a lower ARPP-19 content compared to the values in healthy persons shows a good correlation to the neurodegenerative diseases. The value of healthy people in the respective tissue or in the respective body fluid, which is used in the respective detection set-up, is preferably used as the reference value.

The body fluid is preferably selected from cerebrospinal fluid, spinal cord fluid, blood, serum or nerve extracts. Particularly preferred tissue samples are selected from nerve tissue, preferably brain tissue, in particular nucleus caudatus, nucleus accumbens, putamen, neocortex, cerebrellum, in particular codex cerebrelli, temporal codex or tissue, in particular nerve cells, from peripheral organs.

The degree of phosphorylation of the PKA substrate ARPP-19 is increased by activators of adenylyl cyclase and cAMP. Expression of mRNA encoding ARPP-16/19 was observed in caudate nucleus, putamen, accumbens nucleus and Neocodex. In addition, high levels of ARPP-16/19 were found in the cerebrellar codex. The expression of ARPP-19, however, deviates significantly from the expression of ARPP-16, the ARPP-19 expression levels being highest in the embryo and becoming lower during further development. The 2

AT 410 673 B regional distribution of phosphoproteins in the rat brain has been described using immunohistochemical techniques. In contrast to the specific distribution of ARPP-16, which appears to be restricted to striatal and cortical neurons, ARPP-19 is found in all brain regions and in peripheral organs of the adult rat.

Increased intracellular concentrations of cAMP result in the activation of PKA, which appears to be the main mediator of cAMP responses in mammalian cells and phosphorylates a variety of proteins in nerve cells, including ARPP-19. The mammalian brain is usually a rich source of protein phosphorylation systems. These include various phosphoproteins as substrates, protein kinases, phosphoprotein phosphatases and modulators. A number of phosphoproteins have been shown to function as intracellular messengers in the central nervous system of mammals, and therefore phosphorylation and dephosphorylation of phosphoproteins is an essential mode of regulation of neuronal function. The regulation of the phosphorylation of specific substrate proteins by PKA is an essential mechanism by which neurotransmitters trigger physiological effects in specific target neurons. So far, however, the function of ARPP-19 or other substrates of PKA, especially in connection with newly rodegenerative diseases in humans, has not been known. It was only in the course of the present invention, for example using the method of the differential display polymerase chain reaction (DD-PCR), that ARPP-19 was regulated to a low level, for example in the temporal cortex of patients with Down syndrome. This finding in connection with further data obtained according to the invention demonstrate the inclusion of at least a certain part of the cAMP signaling in the pathology of neurodegenerative diseases in connection with the activities of PKA in the brain of patients who e.g. suffer from Down syndrome or Alzheimer's disease.

According to a particularly preferred method, Alzheimer's disease is detected as a neurodegenerative disease. Other preferred diseases include Pick's disease, Lewybody disease or Jakob Creutzfeld disease. However, it was shown that the reduction in ARPP-19 correlates generally with neurodegenerative diseases, especially in nerve cells. This demonstrates a general mechanism that is responsible for this effect.

According to the invention, the content of ARPP-19 is preferably determined in more than one tissue sample or in more than one body fluid in order to give the diagnosis according to the invention even better support or to be able to produce a more detailed differential diagnosis. The localization of the neurodegenerative diseases is often restricted to certain tissues or - depending on the respective stage of the disease - is different in different tissues, so that this preferred embodiment can also provide essential information on the degree and stage of the disease.

According to a further aspect, the present invention also relates to a kit for the detection of Alzheimer's disease or Down syndrome, which is characterized in that it comprises - molecules for determining ARPP-19 and - a reference value of ARPP-19.

The molecules for the determination of ARPP-19 are not critical and are readily available to the person skilled in the art. Nucleic acid amplification molecules, in particular molecules for the amplification of ARPP-19 mRNA or antibodies against ARPP-19, in particular monoclonal antibodies, are particularly preferred, which are either readily available by the person skilled in the art or can be prepared in a simple manner by the person skilled in the art.

Antibodies of this type can be provided with further detection means which also belong to the prior art (dyes, secondary antibodies, secondary antibody identification parts, affinity group groups which enable binding to magnetic particles, etc.). Various nucleic acid amplification methods, such as PCR and similar methods, are also available for the detection of the ARPP-19 mRNA. Knowledge of the sequence or at least partial sequences or homologies of the ARPP-19 to be determined is a prerequisite for using these nucleic acid amplification methods.

The kit according to the invention can of course also be designed as a multiplex kit for determining the concentration of ARPP-19 and possibly other markers. The conception and 3

AT 41 0 673 B

However, optimization of such tests is easily possible. Tests based on chip technology are particularly preferred.

A sample from a normal pool, preferably in a lyophilized form, is preferably provided in the kit as a reference value, since fluctuations in the standard range are thereby suppressed and differences between individual reference batches can be limited.

According to a further aspect, the present invention relates to the use of ARPP-19 for the detection of Alzheimer's disease or Down syndrome or the use of ARPP-19 to provide detection methods for these neurodegenerative diseases.

The invention is explained in more detail with reference to the following examples and the drawing figure, to which it should of course not be restricted.

It shows:

1: a sequence arrangement of the 157 base cDNA fragment which was found by means of DD-PCR with the sequence of EMBL. The sequence of the 157 base cDNA fragment has 100% homology with the ARPP-19 mRNA sequence.

Examples:

Example 1: Decreased ARPP-19 and PKA levels in brains from Down syndrome and Alzheimer's disease.

MATERIALS AND METHODS

Postmortem samples of human brain (temporal cortex and cerebellum) were obtained from the MRC London Brain Bank for Neurodegenerative Diseases, Institute of Psychiatry. All patients with Down syndrome were caryotyped and had trisomy 21. A formal cognitive assessment of dementia in Down syndrome was not performed. All Down syndrome brains had abundant and extensive beta-amyloid deposits, neurofibril entanglements, and neuritic plaques. Alzheimer's patients met the criteria established by the National Institute of Neurological Disorders and Stroke and Alzheimer's disease and Related Disorders Association for suspected Alzheimer's disease (Tierney et al., Neurology 38 (1989), 359-364). The neuropathological diagnosis " certain Alzheimer's disease " was confirmed using the CERAD criteria (Mirra et al., Neurology 41 (1991), 479-486). Normal brains from individuals who had no neurological or psychiatric history were used as controls. The main cause of death was bronchopneumonia in Down syndrome and Alzheimer's disease and heart disease in the controls. The fresh brain was dissected, corona-shaped sections were snap frozen and stored at -70 ° C until use.

Brain tissue (temporal cortex) was ground using a mortar and pestle under liquid nitrogen and lysed in RNAzol B (Molecular Research Center, Newark, USA). The homogenates, to which 0.1 volume of chloroform had been added, were shaken vigorously for 15 seconds, stored on ice for 5 minutes and centrifuged at 12,000 g for 15 minutes at 4 ° C. Total RNA remained in the upper aqueous phase and was precipitated by shaking with an equal volume of isopropanol. The mixtures were incubated at 4 ° C for 15 minutes and centrifuged at 12,000 g for 15 minutes at 4 ° C. Total RNA pellets were washed with 75% ethanol, dried and dissolved in RNase-free water. To prevent contamination of the chromosome DNA, mRNA was isolated using the Quick Prep Micro mRNA purification kit (Pharmacia Biotech, Uppsala, Sweden), which contains oligo (dT) cellulose, according to the manufacturer's instructions. The concentrations of total RNA and mRNA were calculated spectrophotometrically with an absorption at 260 nm.

A differentiated display polymerase chain reaction (DD-PCR) was carried out using the RNAimage kit (GenHunter Corp., Nashville, USA). Briefly, the isolated mRNA (100ng) was in reverse transcriptase buffer (25mM Tris-Cl, pH 8.3, 37.6mM KCl, 1.5mg 4

AT 410 673 B

MgCI2 and 5 mM DTT), containing 5 units / μΙ MMLV reverse transcriptase, 20 μΜ dNTP mixture and 0.2 μΜ of each single-base-anchored oligo (dT) primer (-G, -A or -C) reverse transcribed. The reverse transcription mixture was used for the PCR at a 1:10 dilution. Thereafter, the PCR (20 μΙ) in PCR buffer (10 mM Tris-Cl, pH 8.4, 50 mM KCl 5, 1.5 mM MgCl 2 and 0.001% gelatin), containing 2 μΜ dNTP, 0.2 μΜ of basebase -verankertem

Oligo (dT) primer, 0.2 μΜ any primer, 0.2 μΙ a- [33P] dATP (2000 Ci / mmol) and 0.05 unit / μΙ AmpliTaq DNA polymerase (Perkin-Elmer) performed. The thermal cycler (GeneAmp PCR System 9700, Perkin-Elmer) was programmed as follows: 40 cycles at 94 ° C for 30 seconds, at 40 ° C for 2 minutes and 72 ° C for 30 seconds, and it ended with 10 a last extension at 72 ° C for 5 minutes. PCR products labeled with 33P were separated on 6% denaturing polyacrylamide gel for 3.5 hours at a constant current of 60 watts. The blotted gel on a piece of 3M paper was dried under vacuum at 80 ° C for 1 hour. The autoradiogram aligned with the dried gel was exposed and developed. The bands of interest were cut away from the dry gels, eluted by boiling in water, and reamplified by PCR with the same set of primers under the same conditions. The reamplified cDNA fragments were cloned into PCR-TRAP vector using the PCR-TRAP cloning system (GenHunter) according to the manufacturer's instructions. DNA sequencing was performed on MWG-BIOTECH (Ebersberg, Germany) and the sequence was compared with all EMBL sequence databases.

Brain tissues were dissolved in homogenate buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (v / v) Tween 20, 4% CHAPS, 1 mM EDTA and a mixture of protease inhibitors, 1 mM PMSF and 1 pg / ml each of pepstatin A, chymostatin, leupeptin and antipain suspended for immunoblotting. The suspension was homogenized in a Potter-Elvehjem homogenizer at 25 4 ° C and centrifuged at 10,000 x g for 10 minutes. The concentration of the protein in the supernatant was determined using the BCA protein test kit (Pierce, USA). Samples (10 µg) were mixed with the same volume of sample buffer (125.5 mM Tris, 70 mM SDS, 0.001% bromophenol blue, 20% glycerol, 2% 2-mercaptoethanol, pH 6.8), incubated for 15 minutes at 95 ° C and loaded onto a homogeneous 12.5% ExcelGel SDS gel (Amersham Pharmacia Biotech, 30 Sweden). The electrophoresis was carried out with Multiphor II Electrophoresis System (Amersham Pharmacia Biotech, Sweden). Proteins separated on the gel were transferred to a PVDF membrane (Millipore, Bedford, USA) and the membrane was placed in blocking buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 0, 1% MgCI2 and 1% lean dry milk) blocked. The membrane was incubated with 35 rabbit antisera against bovine ARPP-19 at room temperature for 2 hours. This was kindly provided by P. Greengard (Laboratory of Molecular and Cellular Neuroscience, Rockefeiler University, NY). After washing three times for 15 minutes with blocking buffer, the membranes were probed with a 1: 1000 dilution of a secondary antibody anti-rabbit IgG (H + L) coupled to horseradish peroxidase (Southern Biotechnology Associates, Inc., Alaba-40 ma , USA) examined for 1 hour. The membrane was washed three times for 15 minutes and developed with the Western Blot Chemiluminescent Reagent (NEN ™ Life Science Products, Inc., USA). The densities of the signaling bands were measured using the RFLP-Scan version 2.1 software program (Scanalytics, USA).

To determine the PKA activities, aliquots of brain samples were homogenized using ultrasound in an ice bath in nine volumes of 5 mM trisacetate buffer (pH 7.4) containing 10 mM mercapto-ethanol. The 10,000 x g supernatant of the homogenate was used to measure enzyme activity. The SpinZyme non-radioactive PKA assay kit (Pierce) was used in accordance with the manufacturer's instructions. Briefly, the samples were incubated with a reaction mixture containing the peptide substrate that had been labeled with a fluorescent dye. The reaction mixture was applied to a separation unit containing the affinity membrane. The phosphorylated product was specifically bound to the membrane, whereas unreacted substrate passed through the support when washed with binding buffer using centrifugation. After the container containing the membrane was transferred to a new receiving device, the bound 55 phosphorylated substrate was eluted from the membrane with elution buffer. The quantification of 5

AT 410 673 B both non-phosphorylated and phosphorylated substrate was carried out by measuring the absorption at 570 nm and the fluorescence readings with an excitation at 573 nm and emission at 589 nm.

The differences between the groups were calculated by the Mann-Whitney U non-parametric U-test using the GraphPad Instat2 program and the level of significance was estimated to be P <0.05. All results are presented as mean ± standard deviation (SA).

RESULTS

In order to detect degenerate genes in the human brain in neurodegenerative diseases, the mRNA expression pattern of the temporal region of patients with Down syndrome and Alzheimer's disease was examined in comparison to controls using DD-PCR. DD-PCR was performed using a total of 24 primer combinations in total. In DD-PCR, a differentially expressed 157 base cDNA fragment was detected, which was determined by a single-base-anchored oligo (dT) primer (5-AAGCT1111I I1111A-3 ') and an arbitrary primer (5'-AAGCTTCGACTGT-3 ') has been amplified. The cDNA bands that were absent from Down syndrome but were expressed in Alzheimer's disease were excised from the gel, amplified again, subcloned and then sequenced. Alignment of the nucleic acid sequence with the sequence obtained from EMBL showed that a 157 base cDNA fragment had 100% homology with the ARPP-19 mRNA sequence (FIG. 1). In the parietal cortex of Down syndrome and Alzheimer's disease, the low expression level of cDNA fragment with a high homology to ARPP-19 mRNA was observed using DD-PCR with the same primers and under the same conditions (the data are not shown). The protein amounts of ARPP-19 in the temporal cortex and in the cerebellum were examined. The protein levels of ARPP-19 were significantly reduced in Down syndrome (temporal cortex) and in Alzheimer's disease (in the cerebellum) as shown in Table 1.

Table 1: ARPP-19 protein levels in brain regions of patients with Down syndrome, Alzheimer's disease and controls.

Control Down syndrome Mzheimer's disease temporal cortex ARPP-19 5.62 ± 2.28 1.36 ± 1.14 * 5.88 ± 4.15 N 6 3 4 age (years) 39.17 ± 16.11 57, 33 ± 11.50 60.25 ± 6.95 postmortem interval (hours) 33.67 + 12.26 44.33 ± 23.97 18.25 ± 17.17 cerebellum ARPP-19 3.12 ± 3.04 2 , 22 ± 2.78 0.83 ± 0.59 * N 6 4 5 age (years) 62.00 ± 9.84 58.50 ± 10.47 60.80 ± 6.83 postmortem interval (hours) 34, 17 ± 12.70 30.00 ± 29.70 17.20 ± 8.38 * P <0.05

The age and postmortem interval of the brain samples used for immunoblotting was different, but was neither between the age nor the postmortem interval and 6

AT 410 673 B the ARPP-19 protein amounts show a correlation (data not specified). The data obtained from the immunoblotting confirmed the "differential display" findings of deteriorated levels of ARPP-19 mRNA in the sleeping cortex of Down syndrome. Since ARPP-19 is a substrate for PKA, the PKA activities in both regions were also determined. PKA activities were reduced in the sleeping cortex from Down syndrome and Alzheimer's disease and in the cerebellum from Down syndrome (Table 2).

Table 2: PKA activities in brain regions of patients with Down syndrome, Alzheimer's disease and controls.

Control Down syndrome Alzheimer's disease cortex 0.55 ± 0.13 0.26 ± 0.09 *** 0.36 ± 0.17 * cerebellum 0.35 ± 0.05 0.20 ± 0.08 ** 0.38 ± 0.09 * N 10 9 9 age (years) 45.10 ± 19.05 55.67 ± 7.48 59.00 ± 8.52 postmortem interval (hours) 29.70 + 11.95 31 , 44 + 19.56 27.11 ± 19.70 *** P <0.001; ** P <0.01; * P &lt; 0.05

The age and postmortem interval of the brain samples had no influence on the PKA activities (data not shown).

DISCUSSION

Significantly reduced protein levels of ARPP-19 were observed in the temporal cortex of Down syndrome, which confirmed the DD-PCR results, which showed worsened mRNA levels of ARPP-19. The significantly reduced protein levels of ARPP-19 were also observed in the cerebellum of Alzheimer's disease. ARPP-19, found in all brain regions, is one of the cAMP-regulated phosphoproteins, such as ARPP-16, ARPP-21, and DARPP-32 (dopamine and cAMP-regulated phosphoprotein). Intracellular cAMP plays key roles as second messenger signals in the modulation of numerous physiological processes. Because the synthesis of cAMP is regulated by adenylylcyclase, which acts with other extracellular stimuli through cell surface receptors coupled to G protein, the amount of cAMP is determined by the activities of adenylylcyclase. Vasoactive intestinal peptide (VIP) and forskolin as activators of adenylate cyclase increased the levels of phosphorylation of ARPP-19. Several studies have reported the involvement of the cAMP signaling pathway in the pathology of Alzheimer's disease and in an animal model of Down syndrome. Basal production of cAMP was found to be impaired and the response of adenylyl cyclase to stimulation of the beta adrenoreceptors with isoprena-lin and the catalytic subunit with forskolin were both in the hippocampus of segmental trisomy (Ts65Dn) mice, a model for mental retardation Down syndrome, greatly reduced. The reduced cAMP signal transduction was reported from the postmortem hippocampus from dementia sufferers in the elderly, showing the significant reduction in both basal and stimulated adenylyl cyclase activity. The markedly reduced activity of adenylyl cyclase was observed in the forebrain and / or sleeping cortex of Alzheimer's disease and was confirmed in the hippocampus and cerebellum of Alzheimer's disease. Reduced levels of brain-specific adenylyl cyclase subtypes have also been reported in patients with Alzheimer's type (DAT) dementia. The immunoreactivities of adenylyl cyclase I and II were significantly reduced, but those of adenylyl cyclase IV and V / VI were unchanged in the parietal cortex membranes at DAT. 7

Claims (8)

AT 410 673 B Reduced amounts of cAMP as a result of the reduced activity of adenylyl cyclase could subsequently lead to reduced PKA activity. PKA, an essential intracellular receptor for cAMP, can mediate the effects of changes in the intracellular concentration of this second messenger and phosphorylate a variety of proteins in nerve tissue. Reduced PKA activity was observed in the temporal cortex in Down syndrome and Alzheimer's disease and in the cerebellum in Down syndrome. Synapsin I, a neuronal phosphoprotein enriched in presynaptic nerve endings, is also regulated by PKA, and in fact, lower levels of Synapsin I have been observed in the Alzheimer hippocampus. Reduced in vitro phosphorylation of synapsin I (site 1) has also been reported in postmortem tissue from Alzheimer's disease. PKA also appears to modulate the function of nuclear factors that bind to DNA sequences that are present in the promoter regions of cAMP-induced genes. Most of these genes contain one or some elements that respond to cAMP, &quot; cAMP-responsive elements &quot; (CREs) and &quot; cAMP response element binding protein &quot; (CREB) is one of them. The significantly reduced immunoreactivity of phosphorylated CREB was reported in DAT. CREB-dependent transcription appears to be essential for long-term memory consolidation in animals. Long-term potentiation of the hippocampus (long-term potentiation, LTP) presumably serves as an elementary mechanism for the creation of certain forms of explicit memory in the mammalian brain. PKA inhibitors blocked a prolonged stage (L-LTP), which is one of the LTP stages and requires protein synthesis. Analogues of cAMP also induced potentiation that blocked naturally induced L-LTP. The activation of presynaptic PKA proved to be necessary for the induction of LTP in the cerebellum. The effect of Alzheimer's amyloid beta peptides on damage to LTP of the hippocampus has been reported, and decreased LTP has also been observed in the Ts65Dn mouse. Based on these studies, the effect of protein phosphorylation on neuronal function could include biochemical events that underlie short-term and long-term memory in neurodegenerative diseases that show dementia. In summary, decreased activity of PKA and its substrate ARPP-19 has been observed in the brains of patients with Down syndrome and Alzheimer's disease. These results suggest that decreased expression of ARPP-19 may be related to decreased PKA activities, although decreased PKA could be a result of decreased levels of cAMP caused by adenylyl cyclase malfunction in neurodegenerative diseases. Alternatively, decreased ARPP-19 may very well be a result of decreased PKA, a guess less likely when viewed in conjunction with the results obtained. In addition, these results may indicate the contribution of ARPP-19 and PKA to faulty phosphorylation and signaling, which is relevant for the pathophysiology of both diseases. These results show that decreased levels of ARPP-19, along with decreased activity of PKA, can lead to a damaged mechanism of cAMP-related signaling and phosphorylation and play a role in the pathophysiology of neurodegenerative diseases. PATENT CLAIMS: 1. A method for the detection of Alzheimer's disease or Down syndrome characterized by the following steps: - Providing a tissue sample or a body fluid of a person with suspected Alzheimer's disease or Down syndrome. - Determining the content of the cAMP-regulated phosphoprotein with a molecular weight of 19,000 (ARPP-19) in the tissue sample or in the body fluid and - Comparing the determined ARPP-19 content with a reference value with which the determination of the presence of Alzheimer's Disease or Down syndrome is possible in this person. 2. The method according to claim 1, characterized in that the value of 8 AT 410 673 B healthy people in the respective tissue or in the respective body fluid is used as a reference value. 3. The method according to claim 1 or 2, characterized in that the body fluid is selected from cerebrospinal fluid, spinal fluid, blood, serum or nerve extracts. 4. The method according to claim 1 or 2, characterized in that the tissue sample is selected from nerve tissue, preferably brain tissue, in particular nucleus cau-datus, nucleus accumbens, putamen, neocortex, cerebrellum, in particular cortex cere-brelli, temporal cortex, tissue, in particular Nerve cells, from peripheral organs. 5. The method according to any one of claims 1 to 4, characterized in that the content of ARPP-19 is determined in more than one tissue sample or in more than one body fluid. 6. Kit for the detection of Alzheimer's disease or Down syndrome, characterized in that it comprises - molecules for the determination of ARPP-19 and - a reference value of ARPP-19. 7. Kit according to claim 6, characterized in that the molecules for the determination of ARPP-19 are selected from nucleic acid amplification molecules, in particular molecules for the amplification of ARPP-19 mRNA, antibodies against ARPP-19, in particular monoclonal antibodies, or Mixtures of these. 8. Kit according to claim 6 or 7, characterized in that a sample from a normal pool, preferably in lyophilized form, is provided as a reference value. THEREFORE 1 SHEET OF DRAWINGS 9