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  • G01N2333/4709—Amyloid plaque core protein

Definitions

• Alzheimer's disease is the most common form of dementia in elderly people. It is a degenerative disease clinically characterised by the progressive decline of cognitive functions, and neuropathologically characterised by the accumulation of insoluble aggregates of ⁇ -amyloid (A ⁇ ) and tau protein, in the cerebral cortex and in subcortical grey matter.
• a ⁇ is deposited in the form of extracellular amyloid in the neuropile (senile plaque) and in the cerebral vessels (congophilic angiopathy), while the tau form of the protein forms anomalous intraneuronal filaments (neurofibrillary degenerations) ⁇ Love S. Neuropathological investigation of dementia: a guide for neurologists. J Neurol Neurosurg Psychiatry 76, Suppl 5:v8-14, 2005) ( Figure 1).
• Alzheimer's disease is sporadic, while in about 5% of the cases it has a familial character and is associated with mutations of 3 genes: presenilin 1 (PSEN 1) on chromosome 14, presenilin 2 (PSEN2) on chromosome 1 and precursor of the ⁇ - amyloid (APP) on chromosome 21.
• PSEN 1 presenilin 1
• PSEN2 presenilin 2
• APP ⁇ - amyloid
• AD' s ethiopathogenesis is not yet entirely understood, but in the last decade the hypothesis has been increasingly confirmed of the "amyloid cascade" (Wilquet et al. Amyloid-beta precursor protein processing in neurodegeneration.
• the A ⁇ derives from its ⁇ APP precursor through a catabolic pathway called "amyloidogenic pathway" ( Figure 2).
• This pathway provides for the cleavage of the molecule upstream and downstream of the ⁇ -protein by two proteases, the beta-secretase and the gamma-secretase.
• the cutting of the beta-secretase (BACE) generates a long, soluble N-terminal fragment (sAPP ⁇ ) and a C- terminal peptide of 99 amino acids (C99).
• sAPP ⁇ beta-secretase
• C99 C-terminal peptide of 99 amino acids
• a ⁇ l 1-40/42 peptide deposition in Alzheimer's disease and young Down's syndrome brains implication of Alzheimer's disease. Acta Neuropathol 112:163-74, 2006).
• ⁇ APP can encounter an alternative catabolic pathway called "non-amyloidogenic", since the protein is cut by another protease (alpha-secretase) at the residues 16-17 of A ⁇ . The action of the latter enzyme thus precludes the formation of ⁇ -amyloid.
• the amyloid cascade hypothesis is supported by multiple proofs:
• a ⁇ particularly in the "long" 42-residue form, shows a strong tendency to be aggregated in oligomers and to form amyloid fibrils which represent the main constituent of the senile plaques ⁇ Armstrong.
• RA Plaques and tangles and the pathogenesis of Alzheimer's disease. Folia Neuropathol 44: 1-11, 2006);
• a ⁇ especially the 42 amino acid form, is neurotoxic (Butterfield et al. Amyloid beta-peptide (1-42) contributes the oxidative stress and neurodegeneration found in Alzheimer disease brain. Brain Pathol 14:426-32, 2004);
• transgenic mice carriers of the APP gene associated with AD, accumulate ⁇ -amyloid in the central nervous system and show deficits of the behavioural-cognitive sphere which are worsened as a function of age ⁇ Kurt et al. Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes Exp Neurol 171:59-71, 2001);
• the A ⁇ l-42 increases until it constitutes 15-40% f the secreted A ⁇ peptides, while in normal conditions it represents only 5-10% thereof (Rocchi et al. Causative and susceptibility genes for Alzheimer's disease: a review. Brain Res Bull 61:1-24, 2003; Lied et al. Clinical, Pathological, and Biochemical Spectrum of Alzheimer Disease Associated with PS-I Mutations. Am J Geriatr Psychiatry 12: 146-56. 2004).
• the technical task of the present invention is that of providing products and their use for the diagnosis and/or prevention and/or care of human and/or animal pathologies characterised by the anomalous deposition of ⁇ -amyloid substance and/or amyloid-like substance in human and/or animal tissues and/or organs, and a screening method for determining the risk of such pathologies.
• the technical task, as well as other objects according to the present invention, are achieved by means of that revealed in the independent claims reported below.
• the present invention refers to the recent discovery of a new punctiform mutation of the human APP gene.
• the mutation is characterised by the substitution of a Cytosine with a Thymidine at codon 673 of the coding sequence of the human APP gene (D8765), corresponding with the nucleotide 2212 (transition c.2212>T) of the isoform of human APP770 ⁇ NMJ00484.2) according to the nomenclature of the GenBank database, accessibly on the website http://www.ncbi.nlm.nih.gov.
• amyloid-like substances it is intended protein aggregates of A ⁇ which do not have the tinctorial and/or ultrastructural characteristics of the amyloid itself.
• Such mutation which induces in the protein sequence the substitution of an alanine with a valine in position 673 (Ala673Val) of APP770, corresponding with the amino acid residue 2 of A ⁇ , was identified in homozygosis of a patient affected with a grave form of dementia with presenile onset.
• the analysis of the cephalorachidian liquid of the patient showed a considerable diminution of the total tau protein and phosphorylated tau, as is observed in Alzheimer disease.
• the plasma levels of A ⁇ 1-40 and A ⁇ l- 42 are increased with respect to control subjects and also with respect to subjects that bear the same mutation in heterozygosis.
• the fibroblasts obtained from skin biopsy of the patient released, in their culture medium, higher quantities of A ⁇ 1-40 and A ⁇ l-42 with respect to control fibroblasts.
• this data whose details are reported in several of the examples listed below, indicates that the mutation Ala673Val, in homozygosis state, is associated with a dementia that can be described as Alzheimer's disease, and, analogous to other mutations of the APP gene, influences the processing of the APP by increasing the A ⁇ production.
• the mixture composed of equimolar quantities of the two peptides not only aggregates less than the mutated peptide but also less than the wild type peptide on its own.
• This "inhibitory" effect on the amyloidogenesis coincides with the clinical observation that the disease is exclusively manifested in the homozygote subjects for the Ala673Val mutation while the heterozygotes, which co-express both the peptides at the cellular level (wild-type and mutated), do not fall ill.
• the heterozygote individuals can be protected from Alzheimer's disease due to the small fibrillogenic tendency of the mutated A ⁇ peptide in the presence of its corresponding wild-type.
• a first application of our invention consists of the production, according to methods known by those skilled in the art, of a vector containing the cDNA of the human APP with Ala673Val, and the use of said vector in order to transfect cell lines usable for pathogenesis studies and therapy.
• a second application consists of the use of the construct according to the previous application as vector for the production, according to methods known by those skilled in the art, of transgenic non- human mammals capable of expressing human APP with Ala673Val mutation, as single form of APP (homozygote animals) or in combination with wild type human APP or containing another mutation (double transgenic).
• Such animals can be used as models for the study of pathogenesis, diagnosis, prevention and care of human and/or animal pathologies, characterised by the anomalous formation and deposition of ⁇ -amyloid and/or amyloid-like substance in organs and tissues.
• the preferred animal is the mouse, and in particular the knockout murine strain C57BL6 for the endogenous APP, and the preferred pathology is AD.
• a construct containing APP with Ala673Val mutation in the in vivo gene therapy (the DNA is transferred directly in the cells or tissues of the patient) or ex vivo gene therapy (the DNA is first transferred in cells isolated from the organism and laboratory- grown, which, thus modified, can be re-introduced in the patient) of pathologies characterised by anomalous deposition of ⁇ -amyloid substance in tissues and organs.
• the transfer of the construct into the target cells can be achieved by means of vectors of viral type, such as for example (a) retroviruses which have the capacity to integrate their DNA inside the proliferation cell chromosomes, (b) lentiviruses which allow transferring genetic material also in cells which do not proliferate, (c) adeno-associated viruses which do not integrate their DNA in the chromosomes of the cell but can be used only for genes of small size, (d) adenoviruses which can transport genes of large size but nevertheless ensure their expression for limited time periods, or (e) herpex simplex virus which only infects several types of cells, in particular the neurons.
• vectors of viral type such as for example (a) retroviruses which have the capacity to integrate their DNA inside the proliferation cell chromosomes, (b) lentiviruses which allow transferring genetic material also in cells which do not proliferate, (c) adeno-associated viruses which do not integrate their DNA in the chromosomes of the cell but can be
• RNA interference RNAi
• RNAi technology applied to our invention can also be useful in the study of the pathogenesis of diseases characterised by anomalous formation and deposition of ⁇ -amyloid and/or amyloid-like substances in the tissues and organs.
• Another application of our invention provides for the use of the human APP with Ala673Val mutation and natural or synthesis peptides containing the mutation itself for the diagnosis, prevention and care of human and/or animal pathologies, characterised by anomalous formation and deposition of ⁇ -amyloid and/or amyloid- like substances in the tissues and organs.
• Our preferred embodiment provides for the use of low molecular weight peptides, like the hexapeptide DVEFRH, suitably formulated for the oral and/or parenteral administration, including the intrathecal administration.
• the preferred pathology is AD.
• the treatment provides for the administration of single peptides or the association of several peptides, used as single treatment or in association with other drugs.
• a further application of our invention provides for the production, by means of techniques known to those skilled in the art, of antibodies towards the proteins and/or peptides pursuant to the previous application, to be used in the diagnosis, prevention and/or care of the of human and/or animal pathologies, characterised by anomalous formation and deposition of ⁇ -amyloid and/or amyloid- like substances in the tissues and organs.
• Our preferred embodiment provides for a monoclonal antibody capable of recognising the Ala673Val mutation in the human APP and in peptides derived therefrom and containing such mutation.
• Such antibody can be used for diagnostic purposes in order to recognise the APP with Ala673Val mutation or, suitably formulated, for the treatment of amyloidosis characterised by the presence of this mutated APP.
• the preferred amyloidosis is AD.
• Example 1 Identification of a new mutation of the APP gene and description of the clinical phenotype of the carrier patient of such mutation.
• the identification of the mutation was conducted by means of the extraction of the genome DNA from the patient lymphocytes, amplification of the exons 16 and 17 of the gene APP by means of polymerase chain reaction (PCR), using the primers 5'- GTTTTGGGTAGCCTTTG-3 and 5'-
• the Ala673Val mutation was identified in homozygosis in a patient without familiality for dementia, affected by an evolutive psycho-organic syndrome with onset at age 36, with ingravescent memory deficits, planning difficulties and behavioural disturbances (Figure 4, III 18).
• the clinical description evolved towards a serious multi-sector cognitive decay, to which involuntary movements are associated of myoclonic type, Parkinsonism and spastic tetraparesis.
• Example 2 Analysis of the chemical-physical characteristics ofA ⁇ peptides containing the Ala673Val mutation. In order to ascertain the effects of the Ala673Val mutation and verify its role in the pathogenesis of AD, we synthesised 2 A ⁇ -40 peptides, one with the wild-type
• DVEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV V sequence and the other containing alanine>valine in position 2 (DVEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV V).
• the peptides were produced by means of solid phase synthesis by using a synthesiser 433A (Applied Biosystems).
• the peptides bonded to the resin were then derivatised at the N-terminal with a lipophile probe (4-dodecylaminocarbonylfluorene-9- ylmethylsuccinimidyl carbonate) according to the method described by Ball et al.
• the peptides wild-type A ⁇ l-40, mutated A ⁇ l- 40 and samples containing equimolar mixtures of the two were dissolved in 10 mM NaOH and subsequently diluted in 50 mM Tris HCl, pH 7.0, at the final concentration of 0.25 and 0.125 mM. The samples were then incubated at 37°C for 1, 4, 8, 24 hours and 3, 5, 10, 15 and 20 days. For each time, aliquots of the samples were analysed in order to determine the secondary structure, the aggregation, the ultrastructure and the optical-tinctorial properties of the aggregate.
• the variations induced by the mutation of the secondary structure of A ⁇ were investigated by means of Circular Dichroism according to the technique described by Clippingdale et al. (JPept Sci 5:227- 49, 2001).
• the peptides were diluted in 150 mM phosphate buffer, pH 7.4, to the final concentration of 100 ⁇ M, and the measurements were conducted with a Jasco-810 spectropolarimeter at a constant 37 0 C temperature.
• the spectra were acquired by using a 1 mm test tube and a scanning speed of 20 nm/min. After having obtained the spectrum of the buffer solution, the noise was reduced, when required, by using the moving average method.
• the aggregation of wild-type A ⁇ l-40, mutated A ⁇ l-40 and their equimolar mixture was evaluated by determining the quantity of peptide that could be sedimented with centrifugation. At the different incubation times, 30 ⁇ l aliquots of the samples were centrifiiged at 15,000 g for 15 minutes at 4°C. The pellet was solubilised in 25 ⁇ l of pure formic acid, and the solution was injected in HPLC provided with PRLP-S IOOA column, 4.6 x 150 mm (Labservice Analytica, Polymer Laboratories).
• the elution was made by using as movable phase an eluent A composed of 0.1% TFA in water and an eluent B constituted by 0.08% TFA in acetonitrile, at a flow speed of 0.7 ml/min, applying a 15-60% linear gradient of the eluent B in 20 min.
• the peak corresponding to the peptide was modified by measuring the absorbance of the eluate at 214 nm.
• the quantity of peptide that can be sedimented was calculated as percentage of the total quantity of peptide present in the initial solution.
• a ⁇ l-40, mutated A ⁇ l-40 and their equimolar mixture were drawn at incubation times in the range of 1 hour - 20 days, deposited on nickel screen covered with Formvar-Carbon for 5 minutes, negatively coloured with an over-saturated solution of uranyl and observed under electronic microscope (EM 109 Zeiss).
• EM 109 Zeiss electronic microscope
• aliquots of the samples were centrifuged at 15,000 g for 15 minutes. The pellets thus obtained were fixed in 2.5% glutaraldehyde in phosphate buffer, pH 7.4, post-fixed in 1% osmium tetroxide, dehydrated in acetone and included in epoxy rein (Spurr, Electron Microscopy Sciences). Ultrafine sections (500 A) were collected on copper screens, coloured with uranyl acetate and lead citrate and observed under the electronic microscope.
• the ultrastructural analysis showed that in the first two days of incubation, the wild-type A ⁇ l-40 peptide forms amorphous aggregates, oligomers and rare filamentous structures. After 48 hours, a short fibril material appears, not ramified, irregular (protofibril), and only after 72 hours of incubation are long rectilinear fibrils observed, of about 8 nm diameters, interposed with amorphous and protofibril material. Subsequently, the density of the fibrils increases and the quantity of amorphous and protofibril material is proportionally increased. Only after 15 days of incubation is most of the material composed of dense fibril networks.
• Example 4 Transfection of cell lines with wild-type human APP, or containing the Ala> VaI mutation in position 2 ofA ⁇ .
• Genetic engineering methods Tesco et al. APP substitutions V715F and L720P alter PSl conformation and differentially affect A ⁇ and AICD generation. J Neurochem 95: 446-56, 2005; Sudhir et al. Release of Amino-terminal Fragments from Amyloid Precursor Protein Reporter and Mutated Derivatives in Cultured Cell. J Biol Chem 267:25602-08, 1992) two vectors were generated respectively containing the cDNA of wild-type human APP751 and the cDNA of human APP751 with the Ala>Val mutation in position 2 of A ⁇ .
• constructs thus produced were further amplified by means of transformation of Top Ten One Shot (Invitrogen) cells, purified by means of the kit Endofree Plasmid Maxi Kit (Qiagen), and used for transfecting COS7 and CHO cells by means of electroporation.
• the efficiency of the transfections was evaluated through the quantification of APP on cell lysates by means of Western blot, using the antibody 22Cl 1 (Chernicon International Inc.) directed against the N-terminal region of the protein (residues 61-88).
• the APP expression level was used for comparing the levels of A ⁇ production by cells transfected with two constructs.
• the metering was then carried out of peptides A ⁇ l-40, A ⁇ l-42 and truncated forms at the N-terminal with ELISA (Immuno-Biological Laboratories Gunma). The study demonstrated:
• Example 5 Generation of transgenic mice, carriers of the Ala> Val mutation in position 2 ofA ⁇ .
• the cDNA of wild-type APP751 was cloned in the vector pTSC21, containing the promoter murine Thy 1.2 (restriction sites HindIII and EcoRV) ( Figure 18).
• the construct was then subjected to site- specific mutagenesis with insertion of the Ala>Val mutation in position 2 of A ⁇ (Stratagene) by means of the same protocol reported for the cell transfections (see Example 4), and it was used for generating transgenic mice starting from the strain C57BI/6. 6 founders (3 male and 3 female) positive for the transgene were obtained, which gave life to three lines which over-express human APP with Ala>Val mutation in position 2 of A ⁇ in the central nervous system.
• mice expressing human APP with mutation 2 of A ⁇ in homozygosis and heterozygosis will be used for pathogenesis studies, diagnosis, prevention and care of Alzheimer's disease and, more in general, of human and/or animal diseases characterised by an anomalous deposition of amyloid and/or amyloid-like substance in organs and tissues.
• AD Alzheimer's disease
• AICD C-terminal fragments which derives from the cutting of
• APP 673V APP with Ala>Val mutation at codon 673
• a ⁇ ⁇ -amyloid, peptide deriving from the catabolism of APP
• COS Cells kidney cells of adult male Cercopithecus aethiops transformed with a defective mutant of the SV40 virus
• Cellule CHO Cells derived from Chinese hamster ovary
• DHPLC Denaturing high performance liquid chromatography
• DNA deoxyribonucleic acid
• FAD Familial form of Alzheimer's disease
• HPLC High performance liquid chromatography
• RNA ribonucleic acid

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  • 2008-10-10 JP JP2010528323A patent/JP2011500007A/ja active Pending

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