Patent Application
20180057552
The present invention relates to a peptide or collection of peptides derived from amyloid precursor protein (APP). The present invention also relates to uses of such peptide or collection of peptides, in particular as a diagnostic marker and/or as a drug target.
Intensive efforts are on the way to develop therapeutic strategies targeting Aβ, the major component of the Alzheimer's disease signifying amyloid plaques1. Aβ is generated from APP by proteolytic processing. First, β-secretase, identified as BACE1, cleaves at the Met-Asp bond of the Aβ domain and generates CTF-β, which is further processed to Aβ by intramembrane cleavage by γ-secretase6. Liberated Aβ forms soluble synaptotoxic oligomers, which are believed to be the major culprit of the disease1. Thus targeting the production of synaptotoxic Aβ species by β-secretase inhibition is a promising therapeutic strategy and clinical studies with high affinity BACE1 inhibitors are currently evaluated7,8. Decreasing BACE1 activity leads to an increase of non-amyloidogenic processing via ADAM109. Similarly, enhancing α-secretase activity reduced Aβ production and plaque formation10. However, stable isotope labeling kinetics upon in vivo inhibition of BACE1 in monkeys revealed an 83% decrease of sAPP-β but only a 35% increase of sAPP-α3. Thus, the fate of almost 50% of the initially labeled APP remains unclear. In addition to the two major and well-studied proteolytic processing pathways, APP is also shed in minor processing pathways utilizing different proteases11. Furthermore, 17-35 kDa N-APP fragments are generated during early development and upon trophic-factor deprivation12-14. However, such alternative APP metabolites were not observed to accumulate upon BACE1 inhibition.
Hence, there is a need in the art to identify new pathways that are involved in the genesis of Alzheimer's disease. More specifically, there is a need in the art to identify molecular species which may either serve as a diagnostic marker or a drug target, more specifically, as a diagnostic marker or drug target for Alzheimer's disease.
In a first aspect, the present invention relates to a peptide or collection of peptides derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP, and by the (further) action of at least one enzyme selected from α-secretase (ADAM10) and β-secretase (β-site APP cleaving enzyme 1; BACE1).
In one embodiment, the peptide or collection of peptides according to the present invention is derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP, and both α-secretase (ADAM10) and β-secretase (β-site APP cleaving enzyme 1; BACE1).
In one embodiment, the peptide or collection of peptides has one or several epitopes selected from MISEPRISYG (SEQ ID NO: 1), DALMPSLT (SEQ ID NO: 2), PWHSFGADSVP (SEQ ID NO: 3), SEVKM (SEQ ID NO: 4), and DAEFRHDSGYEVHHQK (SEQ ID NO: 5).
In one embodiment, the peptide or collection of peptides is characterized by recognition through an antibody which recognizes one or several of said aforementioned epitopes.
In one embodiment, the peptide or collection of peptides has a sequence selected from MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM (SEQ ID NO: 6) and MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQK (SEQ ID NO: 7).
In one embodiment, one peptide has a sequence MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM (SEQ ID NO: 6) and another peptide has a sequence MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQK (SEQ ID NO: 7).
In a further aspect, the present invention relates to a composition of peptides comprising one or several peptides according to the present invention and at least one further peptide derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP only.
In one embodiment, the at least one further peptide is membrane bound or is soluble in aqueous solution.
In one embodiment, the composition according to the present invention comprises both a membrane bound further peptide derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP only and a peptide soluble in aqueous solution and derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP only.
In one embodiment, said membrane bound further peptide has a sequence represented by SEQ ID NO: 8, and said peptide soluble in aqueous solution has a sequence represented by SEQ ID NO. 9.
The invention also relates to the peptide or collection of peptides or the composition of peptides according to the present invention, for use as a diagnostic marker.
In one embodiment, said use involves the detection of said peptide or of said collection or composition of peptides in cerebrospinal fluid (CSF) or plasma.
In one embodiment, said diagnostic marker is a marker for Alzheimer's disease.
The present invention furthermore relates to the peptide or collection of peptides or the composition of peptides according to the present invention, for use as a drug target, preferably as a drug target in the treatment, prevention and/or alleviation of Alzheimer's disease.
In one embodiment, said use as a drug target involves the inhibition of formation or action of said peptide or said collection of peptides or of said composition of peptides, or the inhibition of action or production of matrix-metalloproteinase MT5-MMP and/or MT1-MMP.
As used herein, a “peptide derived from amyloid precursor protein (APP) by the action of one or several matrix-metalloproteinases” is meant to refer to a peptide that is the reaction product of the one or several matrix-metalloproteinases acting on the amyloid precursor protein and cleaving the same into fragment(s). The present inventors have surprisingly identified a novel prominent proteolytic pathway which is initiated by the action of matrix-metalloproteinase 5 and/or matrix-metalloproteinase 1 or both. As used herein, the action of such matrix-metalloproteinase 5 and/or 1 is herein also sometimes referred to as “η-secretase”. The present inventors believe that this novel proteolytic pathway is initiated by such η-secretase and is followed by the action of other secretases, most notably α-secretase (ADAM10) and/or β-secretase (β-site APP cleaving enzyme 1; BACE1). The resultant peptides are herein also sometimes referred to as “Aη-peptides” which comprise Aη-β and Aη-β (see also
The present invention also relates to the use of the peptide(s) or collection of peptides according to the present invention for the manufacture of a medicament for the prevention, treatment and/or alleviation of Alzheimer's disease. In one embodiment, said use involves the administration of a compound to a patient, which compound inhibits the formation or action of one or several peptides according to the present invention. Preferably, said patient is a human patient.
The present invention also relates to a method of identifying a compound useful for the prevention, treatment and/or alleviation of Alzheimer's disease, wherein said compound is identified based on its capability to inhibit the formation or action of one or several peptides according to the present invention.
The present invention also relates to a method of detecting Alzheimer's disease in a patient, wherein said method involves detecting one or several peptides in accordance with the present invention, i.e. peptides, derived from amyloid precursor protein (APP) by the action of matrix-metalloproteinase MT1-MMP or MT5-MMP, and by the action of at least one enzyme selected from α-secretase (ADAM10) and β-secretase (β-site APP cleaving enzyme 1; BACE1). In one embodiment, said detection occurs by detecting said peptide(s) in cerebrospinalfluid (CSF) and/or plasma. Preferably said patient is a human patient.
The present invention also relates to a method of treatment, prevention and/or alleviation of Alzheimer's disease, wherein said method involves the inhibition, in a patient, of the formation or the action of the peptide(s) in accordance with the present invention, or it involves the inhibition of the action or production of matrix-metalloproteinase MT5-MMP and/or MT1-MMP. In one embodiment, the inhibition of the formation or action of said peptide(s) is achieved by the administration to a patient of a drug that inhibits the formation of said peptide(s). In another embodiment, said method of treatment involves the administration to a patient of a drug that inhibits the action of said peptide(s) Inhibition of the formation of said peptide(s) may for example occur by binding to APP and/or to the enzyme(s) cleaving said APP. The inhibition of the action of said peptide(s) may occur by binding to said peptide(s) or to its (their) respective binding partner(s). Preferably, said patient is a human patient.
The specific sequences disclosed herein refer to and are derived from the specific splicing variant P05067-4 (as disclosed in the data base UniProt, human isoform APP695 of amyloid beta A4 protein). It should be clear, however, that the peptide(s) according to the present invention are not limited to the peptides derived from this specific splicing variant, and a person skilled in the art will be able to identify corresponding equivalent peptides from other splicing variants as well. It should also be noted that the sequences used herein are human sequences, despite the fact that some of the experiments performed herein were done with mice. However, the data obtained in
Furthermore, reference is made to the figures, wherein
More specifically,
a) Schematic representation of the η-secretase pathway (left) and previously known amyloidogenic pathway (right). Antibodies used in this study are indicated (see also
a) MMPs can cleave human APP695 at the indicated position (arrow) between amino acid N504 and M505 in the N-terminal domain. The epitope for the novel cleavage-specific antibody 10A8 is indicated (grey). sAPP-η of 80 kDa was specifically detected in DEA extracts of P10 WT mouse brain using the antibody 10A8, but was absent in APP KO brains. b-c) Soluble Aη levels, detected by the 9478D and M3.2 antibodies (Aη-α) were reduced in MT5-MMP −/−mouse brains, but unchanged in MT1-MMP−/−brains. Soluble (DEA) brain lysates were prepared from P10 mice. Total levels of secreted APP (22C11), sAPP-α or sAPP-β were unchanged.
a) Supernatants of CHO cells expressing human APPV717F without or with BACE1 inhibition (BI; 2 μM Merck IV) were compared to synthetic peptides of Aη-β and Aη-α. Increased Aη-α peptide levels were observed upon BACE1 inhibitor treatment with 2D8 and 2E9 antibodies. The fragment with the lowest molecular weight, co-migrating with the synthetic peptide Aη-β, disappeared upon BACE1 inhibition b) After overnight incubation of DIV16 primary hippocampal neurons without or with the BACE1 inhibitor (BI; 2 μM Merck IV), supernatants were analyzed by Western blotting. A strong increase of endogenous Aη-α was detected with M3.2 antibody. Total levels of secreted APP (22C11) were unchanged while sAPP-α levels increased. The absence of sAPP-β and Aβ proves the effective blockade of BACE1. c) Western blot analysis of DEA extracts of P10 BACE1−/−mouse brains revealed a significant increase in Aη-α peptides as compared to controls. Total levels of secreted APP (22C11) were unchanged while sAPP-α levels increased due to compensation by α-secretase activity. CTF-η levels were increased in RIPA lysates of the BACE1 KO mouse brain. As expected after an efficient BACE1 block, CTF-β and sAPP-β were severely reduced. d) BACE1 inhibition in vivo resulted in enhanced production of Aη-α species. A schematic presentation of the study design with a single oral dose treatment of APPV7171 mice with the BACE1 inhibitor RO5508887 is shown in the upper panel. Inhibitor treated mice and vehicle treated controls were sacrificed and analyzed after 5, 8 or 24 h. BACE1 inhibition reduced sAPP-β and CTF-β and increased levels of Aη-α at 5 and 8 h after treatment. 24 h after the treatment these changes were normalized due to the clearance of the inhibitor (a background band obtained with Y188 is indicated by asterisk).
a) Aη-α and Aη-β peptides were expressed in CHO cells and collected in OPTIMEM medium. Western blot analysis revealed the larger Aη-α and the smaller Aη-β peptides. Higher molecular weight bands are most likely due to posttranslational modification by glycosylation. b-e) SEC fractions enriched for Aη were diluted (1:15) in ACSF for the treatment of hippocampal slices and LTP measurements. Aη-α, Aη-β and control SEC fractions (obtained from CHO cells transfected with the empty vector) were perfused over mouse hippocampal slices for 15 min after obtaining a stable baseline of a fEPSP at the CA3-CA1 synapse. At the end of these 15 minutes, a high-frequency stimulation protocol was applied (HFS; 2× (100 Hz, 1 s) at 20 second inter-stimulus interval) to induce long-term potentiation (LTP). b) CHO supernatant by itself did not alter LTP when compared to ACSF control. c) SEC fractions from conditioned media of CHO cells expressing Aη-α significantly inhibited LTP. d) SEC fractions from conditioned media of CHO cells expressing Aη-β did not significantly inhibit LTP. e) Quantification of LTP magnitudes (as % of baseline) calculated 45-60 minutes post-HFS from graphs in b-d with statistical analysis (*p<0.05); error bars represent s.e.m. n=number of fields.
List of antibodies used with indicated specificity, antibody epitope (if known) and dilutions applied in Western blotting and immunostaining techniques.
Immunohistochemistry with 22C11 (a, b), 9478D (c, d) and 9476M (e, f) in the human hippocampus (CA1-subiculum region) of a control case (a, c, e) and in the AD case (b, d, f). Immuno-positive signals were observed with 22C11 (a), 9478D (c) and 9476M (e) in the somata and neuropils of normal and AD brain. In AD brains these antibodies decorate dystrophic neurites (b, d, f). Calibration bar=30 μm.
a) Treatment with AβS26C dimers (100 nM final; JPT Peptide Technologies, Germany; diluted in 25 ml re-circulating ACSF) reduced LTP as compared to interleaved LTP recordings in 25 ml re-circulating ACSF. b) Illustrated is the average LTP magnitude (at 45-60 minutes post-LTP induction) normalized to pre-LTP baseline values (100%) in untreated conditions (**p<0.01).
a, Expression of MT1-MMP and MT5-MMP in stably transfected N2a cells were analyzed in lysates with specific antibodies. No major difference was observed in the levels of APP-FL (APP full length) and APP-CTFs, while 2 predominant bands were observed in the range of 20-30 kDa. With antibodies Y188 and with M3.2 an accumulation of CTF-η was detected in both cell lines. While the smaller CTF-η species was enriched in N2a MT1-MMP cells, the higher CTF-η species was enriched in N2a MT5-MMP cells. In lysates of N2a MT1-MMP cells additionally a 16 kDa fragment was identified by both antibodies and as APP-CTF derived from a cleavage closer to the β-secretase cleavage site. b, In supernatants of N2a cells expressing either MT1-MMP or MT5-MMP the levels of sAPP-α and sAPP-β were substantially reduced, while sAPP-η was strongly increased as detected with the antibody 10A8. The antibody M3.2, directed against the murine N-terminal Aβ domain, allowed to specifically detect a longer Aη-α species of 16 kDa in the supernatants of N2a MT5-MMP cells, while a slightly shorter Aη-α species of 14 kDa was observed in the supernatants of N2a MT1-MMP cells. Here additionally a 7 kDa species was detected with the antibody M3.2. While this peptide is most likely derived from the 16 kDa CTF observed in the N2a MT1-MMP lysates, the Aη-α species of higher or lower molecular weight correspond to the species seen in control N2a supernatants and fit to the different sizes found for the CTF-Aη species in the respective cell lysates.
Furthermore, reference is made to the following sequences which are given as exemplary embodiments:
All sequences are Homo sapiens sequences. It should be noted that the sequences indicated in the present application are denoted such that their N-terminal end is at the left side, and their C-terminal end is at the right side. Hence, as an example, the sequence MISEPRISYG has the N-terminal end at M and the C-terminal end at G.
Example of an Aη-β-peptide (residues 505-596 of P05067-4)
Example of an Aη-α-peptide (residues 505-612 of P05067-4)
Example of an CTF-η-peptide (residues 505-695 of P05067-4)
Example of an sAPPη-peptide (residues 19-504 of P05067-4)
Human isoform APP695 of amyloid beta A4 protein (P05067-4)(residues 1-695)
The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies used in the context of the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
Antibodies may be generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term “antibody” should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody peptide, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
Monoclonal antibodies directed against full length or peptide fragments of a peptide or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice or other animals, such as rabbits, hamsters, etc. immunized with the peptide using standard procedures as referenced herein.
Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and the references cited therein. Further, the antibody of the invention may be “humanized” using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759).
To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired peptide to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook and Russell (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).
Bacteriophage which encode the desired antibody, may be engineered such that the peptide is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding peptide, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al., (supra).
Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human antibody fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
Moreover, reference is made to the following examples which are given to illustrate, not to limit the present invention:
Material and Methods
Biochemical Methods
Soluble proteins were extracted from brain hemispheres with DEA buffer39, membrane proteins were extracted with RIPA buffer or by applying a membrane preparation protocol as described40. All Western blot procedures were done essentially as described41.
BACE1 Inhibitor Treatment
Three-month-old heterozygous female transgenic mice FVB/N×C57B1/6J expressing APPV717128 were used for BACE1 inhibition studies. Gavage mediated administration of BACE1 inhibitor RO5508887 (90 mg/kg, 14.06 ml/kg) or vehicle (14.06 ml/kg) was performed once27.
Slice Preparation and Electrophysiological Recordings
OPTIMEM was used to collect CHO cell supernatants. SEC was performed with concentrated CHO supernatants using a FPLC (ÄKTApurifier) equipped with a Superdex75 column (GE Healthcare). 1 ml samples were collected with a mobile phase flow rate set at 0.5 ml/min in standard ACSF. Acute hippocampal slices were prepared from Swiss mice (PND20-30) and kept in ACSF42. Extracellular field excitatory post-synaptic potentials (fEPSPs) were obtained from CA1 pyramidal neurons. After 15 minutes of bath application of SEC fractions43, LTP was induced by high frequency stimulation (2 pulses of 100 Hz for 1 second with a 20 second inter-pulse interval). LTP was recorded for 60 minutes and statistical analysis was performed on the last 15 minutes of recording compared to baseline fEPSP values.
Cell Culture
CHO and 7PA244 cells were grown in DMEM/F12 (Thermo Scientific) supplemented with 10% fetal calf serum (FCS, Thermo Scientific) plus Penicillin/Streptomycin and NEAA (Non-essential amino acids, PAA) in a humid incubator with 5% CO2 at a temperature of 37° C. For inhibitor treatment, cell culture medium was replaced with fresh, pre-warmed serum free medium (OPTIMEM; Thermo Scientific) supplemented with inhibitors or DMSO as vehicle control. Treatment was initiated when cells reached 90-100% confluency and conditioned media were harvested after 20-24 h. Supernatants were cleared by centrifugation (10′, 5500 g at 4° C.). To obtain cell lysates, cell monolayers were washed once with ice-cold PBS and detached in 1 ml PBS using a cell scraper. The cell suspension was pelleted by centrifugation (5′, 1000 g at 4° C.) and lysed with RIPA buffer (20 mM sodium citrate pH 6.4, 1 mM EDTA, 1% Triton X-100 in ddH2O) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich). The protein concentration of lysates was determined using the Uptima BC Assay Protein Quantitation kit (Interchim).
Primary Cell Culture
Hippocampal neurons were isolated from embryonic day 18 CD rats (Charles River) as described45. Dissociated neurons were plated at 17,700 cells per cm2 onto 6 cm dishes coated with poly-L-lysine (1 mg/mL; Sigma) and cultured in Neurobasal medium supplemented with 2% B27 and 0.5 mM L-glutamine (all from Invitrogen). Hippocampal cultures were maintained in a humidified 5% CO2 incubator at 37° C. For inhibitor treatment, DIV16 culture medium was replaced with fresh, pre-equilibrated N2 medium (supplemented with 20% of four days conditioned N2 medium from pure primary cultured astrocytes) to which inhibitors or DMSO as vehicle control were added45.
Transgenic Mice, Animal Care, and Animal Handling
BACE1−/− and APPPS-21 mice were described before46,47 and were bred for this study in a B16C57/J background. All treatments were approved by the local committee for animal use and were performed in accordance to state and federal regulations (license number KVR-I/221-TA116/09). Mice had access to pre-filtered sterile water and standard mouse chow (Ssniff® Ms-H, Ssniff Spezialdiäten GmbH, Soest, Germany) ad libitum and were housed under a reversed day-night rhythm in IVC System Typ II L-cages (528 cm2) equipped with solid floors and a layer of bedding, in accordance to local legislation on animal welfare.
BACE1 Inhibitor Treatment
APPV717148 mice were treated with the inhibitor RO5508887 provided by Hoffmann-La Roche49. Three-month-old heterozygous female transgenic mice in mixed FVB/N×C57B1/6J background expressing hAPPV717148 were used for BACE1 inhibition studies. Gavage mediated administration of BACE1 inhibitor (90 mg/kg, 14.06 ml/kg) or vehicle (14.06 ml/kg) was performed once49. The BACE1 inhibitor was diluted in 5% ethanol (Merck) and 10% solutol (Sigma-Aldrich) in sterile water (Baxter). Animals were sacrificed after 5, 8 and 24 h. Mice were anesthetized with 3.5 μl per gram body weight of a mixture of ketamine (115 mg/ml ketamine hydrochloride, Eurovet), xylazin 2% (23.32 mg/ml xylazine hydrochloride, VMD Arendonk), atropine (0.50 mg/ml atropine sulphate, Sterop) and saline (8:5:2:5, v/v/v/v). For brain preparation, mice were flushed trans-cardially with ice-cold saline (3.5 ml/min, 3 min). The brain was removed from the cranium and dissected into left and right hemiforebrain, brainstem, cerebellum and olfactory bulb. The brain structures were promptly immersed in liquid nitrogen and stored at −80° C. Different tissues (kidneys, spleen, liver, stomach, gut, lungs and heart) were examined and checked for gross abnormalities. No obvious abnormalities were observed in any of the treatment groups.
Preparation of Protein Extracts from Brain
Brains were removed from the cranium and dissected into left and right hemispheres. Brain tissue was snap frozen in liquid nitrogen and stored at −80° C. Soluble proteins were extracted with DEA buffer (50 mM NaCl, 0.2% Diethylamine, pH 10+protease inhibitor (P8340, Sigma-Aldrich)50, membrane proteins were extracted with RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate+protease inhibitor) or applying a membrane preparation protocol as described before51.
Protein Analysis
Proteins were separated under denaturing conditions using discontinuous SDS-PAGE. Equal amounts of proteins denatured in Laemmli buffer were loaded onto the gel and 10 μl of the SeeBlue Plus2 Prestained Standard (Invitrogen) served as molecular weight marker. Electrophoresis was performed in Tris-glycine buffer (25 mM Tris, 190 mM glycine in ddH2O) using the Mini-PROTEAN system (BIORAD) on activated PVDF membranes. Low molecular weight proteins (<16 kDa) were separated using precast gradient Tricine Protein Gels (10-20%, 1 mm, Novex) in Tris-tricine buffer using the XCell SureLock Mini-Cell system (Novex). After separation by SDS-PAGE proteins were transferred onto membranes using the tank/wet Mini Trans-Blot cell system (BIORAD). CTFs, Aη and Aβ were detected after transfer on Nitrocellulose membranes (Protran BA85; GE Healthcare), while other proteins were blotted on PVDF (Immobilon-P, Merck Millipore). As size markers for Aη synthetic peptides Aη-β (92 aa; MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM92) (=SEQ ID NO: 6) and the slightly longer Aη-α (108 aa; MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEV EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQK108) (=SEQ ID NO: 7) were obtained from Peptide Specialty Laboratories (PSL; Heidelberg, Germany). Upon completion of the transfer and prior to blocking, proteins transferred to nitrocellulose membranes were additionally denatured by boiling the membrane in PBS (140 mM NaCl, 10 mM Na2HPO4, 1.75 mM KH2PO4, 2.7 mM KCl in ddH2O, pH 7.4) for 5 min. After cooling to room temperature nitrocellulose membranes as well as the PVDF membranes were blocked in I-Block solution (0.2% Tropix I-Block (Applied Biosystems), 0.1% Tween20 in PBS) for 1 h at room temperature or o/n at 4° C. (with agitation). Transferred proteins were detected using immunodetection and enhanced chemiluminescence (ECL). First, blocked membranes were incubated with primary antibodies diluted in I-Block solution o/n at 4° C. (with agitation). After removal of the antibody, membranes were washed 3× in TBS-T buffer (10 min each, at room temperature, with agitation; 140 mM NaCl, 2.68 mM KCl, 24.76 mM Tris, 0.3% Triton X-100 in ddH2O, pH 7.6) and subsequently incubated with a horseradish peroxidase (HRP) coupled secondary antibody (obtained from Promega or Santa Cruz). Secondary antibodies were diluted in I-Block solution and membranes were incubated for 1 h at RT (with agitation) followed by three washes in TBS-T. For ECL detection, membranes were incubated with HRP substrate (ECL, GE Healthcare or ECL Plus, Thermo Scientific) for 1 min at RT and signals were captured with X-ray films (Super RX Medical X-Ray, Fujifilm), which were subsequently developed using an automated film developer (CAWOMAT 2000 IR, CAWO).
Human CSF Samples
Human CSF samples were collected at the Dept. of Neurology Outpatient unit for neurodegenerative disease (KBFZ) of the University of Bonn. CSF was obtained by lumbar puncture at position L3, centrifuged and divided in small aliquots. For further analysis, samples were stored at −80° C. Turbid or blood contaminated samples were excluded from analysis. Use of these samples for research purposes has been consented by all patients according to the ethical committee requirements of the University of Bonn Ethical committee and approval number 279/10.
Neuropathology & Immunohistochemistry
Braak-NFT stages52, and CERAD53 scores for neuritic plaques were used to determine the degree of AD pathology according to the NIA-AA guidelines54. Consecutive paraffin sections from the human medial lobe were stained with 22C11, 9476M and 9478D Primary antibodies were detected with biotinylated anti-mouse and anti-rabbit IgG secondary antibodies and visualized with avidin-biotin-complex (ABC-Kit, Vector Laboratories) and Diaminobenzidine-HCl (DAB). The sections were counterstained with Haematoxylin. Positive and negative controls were performed. 9476M and 9478D stainings were assessed in 10 control and 10 AD patient cases.
For double immunofluorescence analysis of APPPS-21 brain sections, 6 month old mice were sacrificed by CO2 inhalation according to animal handling laws. Brains were dissected and fixed with 4% paraformaldehyde (PFA) in 0.1M PBS, pH 7.4 for 48 hours. For immunohistochemistry, 25 μm-thick sagittal mouse brain cryosections were treated with 10 mM sodium citrate, pH 6 at 93° C. for 20 minutes, washed with 0.5% Triton X-100 in PBS, blocked with 5% goat serum (Invitrogen) and 0.5% Triton X-100 in PBS for 1 h and subsequently incubated overnight with primary antibodies diluted in blocking solution. Primary antibodies were used as listed in
Laser Capture Microdissection (LCM)
For laser capture microdissection of plaque cores and halos, 14 months old transgenic APPPS-21 mice were used according to a previously published protocol55 with slight modifications. Mice brains were dissected, embedded in Shandon M1 embedding matrix (Thermo Scientific) and immediately frozen on crushed dry ice. 10 μm-thick sagittal sections were cut using a Microm HM 560 cryostat (Thermo Scientific), mounted on frame slides containing a 1.4 μm polyethylene terephthalate (PET)-membrane (Leica Microsystems) and subsequently stained or stored at −80° C. for later usage. Staining was performed as follows: brain sections were thawed shortly at room temperature, fixed with 75% ethanol for 1 min, stained with 0.05% Thioflavin-S for 5 min, washed with 75% ethanol and dried at room temperature. LCM was performed on the same day using a laser dissection microscope (Leica, LMD 7000) with the following settings: excitation wavelength 495 nm, laser power 30, aperture 5, speed 6 and pulse frequency 119. Plaque cores and halos were cut using a 63× magnification objective and control non-plaque areas using a 10× magnification objective and collected in 0.5 ml caps (Leica Microsystem) for protein analysis. Lysates were done essentially as described above using RIPA with 0.1% SDS.
Slice Preparation and Electrophysiological Recordings
Transverse hippocampal slices (350 μm) were prepared from postnatal day 20-30 Swiss mice following standard procedures56. Slices were cut in ice-cold oxygenated (95% O2/5% CO2) solution containing 206 mM sucrose, 2.8 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 1 mM MgCl2, 1 mM CaCl2, 26 mM NaHCO3, 0.4 mM sodium ascorbate, 10 mM glucose (pH 7.4). For recovery (1 h), slices were incubated at 27° C. in oxygenated standard artificial cerebrospinal fluid (ACSF) containing: 124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 3.6 mM CaCl2, 26 mM NaHCO3, 0.4 mM sodium ascorbate, 10 mM glucose (pH 7.4)57. Slices were inspected in a chamber on an upright microscope (Slicescope, Scientifica Ltd) with IR-DIC illumination and were perfused with the oxygenated ACSF at 27±1° C. Field excitatory post-synaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region using a glass electrode (filled with 1 M NaCl/10 mM HEPES, pH 7.4) and the stimuli (30% of maximal (EPSP) were delivered to the Schaeffer Collateral pathway by a monopolar glass electrode (filled with ACSF). Electrodes were specifically placed just below the surface of the slice to maximize the exposure to circulating peptides. A minimum of 15 minutes stable baseline was first obtained in standard ACSF followed by another 15 minutes of bath application of ACSF containing SEC fractions (CHO, Aη-α or Aη-β; 1/15 dilution, interleaved recordings) using re-circulation with a peristaltic pump at 2.5-3 ml min−1 while being continuously aerated with 95% oxygen. No alterations in fEPSP baseline responses were observed after incubation with the SEC fractions. In the continuous presence of the ACSF/SEC solution, LTP was then induced using a high frequency stimulation (HSF) protocol with 2 pulses of 100 Hz for 1 sec with 20 sec interval between pulses, and recorded for one hour. Control recordings (no application of SEC fractions) were obtained in an interleaved fashion where ACSF was re-circulated using an identical procedure. For LTP analysis, the first third of the fEPSP slope was calculated in baseline condition (15 minutes prior to induction of LTP and for 60 minutes post-induction). The average baseline value was normalized to 100% and all values of the experiment were normalized to this baseline average (one minute bins). Experiments were pooled per condition and presented as mean*s.e.m. Data analysis was performed with the clampfit software (Molecular devices). Statistical analysis was performed using GraphPad (Prism 6) on the last fifteen minutes of the recordings vs the 15 minutes of baseline using a one way-ANOVA and post-hoc Bonferroni test.
Results
To identify novel proteolytic processing pathways of APP, the present inventors searched for C-terminal fragments (CTFs) of APP different from those giving rise to p3 (CTF-α) or Aβ (CTF-β) in membrane fractions of mouse brains15-17. Indeed, this revealed a novel CTF with an approximate molecular weight of 30 kDa, recognized by an antibody to the C-terminus of APP (Y188), which is absent in the brains of APP knockout mice (APPKO)18 (
Since membrane bound matrix-metalloproteinases like MT1-MMP and MT5-MMP were shown to cleave in vitro at a site consistent with η-secretase cleavage23,24, the present inventors produced a neo-epitope specific antibody (10A8;
While investigating protease inhibitors capable to modulate η-secretase activity, the present inventors observed that pharmacological BACE1 inhibition led to a pronounced accumulation of the long Aη-α species in CHO cells overexpressing APPV717F (
Since cleavage products of the Aη processing pathway accumulate upon BACE1 inhibition (
The features of the present invention disclosed in the specification, the claims, and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15159877.8 | Mar 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/055081 | 3/10/2016 | WO | 00 |
