ANIMAL MODEL FOR NEURODEGENERATIVE DISORDERS
20220361462 · 2022-11-17
Inventors
Cpc classification
A61K49/0008
HUMAN NECESSITIES
A01K2207/10
HUMAN NECESSITIES
International classification
Abstract
The invention relates to animal models, and in particular to novel in vivo animal models for neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease or Motor Neurone Disease. The invention extends to methods for providing such models. The invention also provides animal models per se and methods for investigating the underlying mechanisms occurring in such neurodegenerative disorders, in particular, Alzheimer's disease, and also extends to models, methods and assays for testing pharmacological test compounds, which may modulate neurological processes, and for drug screening for use in treating neurodegenerative diseases.
Claims
1. A method of providing an animal model for a neurodegenerative disease, the method comprising introducing, into the brain of a non-human animal, a peptide comprising or consisting of the amino acid sequence represented as SEQ ID NO: 3, or an active variant of fragment thereof, wherein the peptide causes an increase in Tau protein in one or more sites in the animal's brain.
2. A method according to claim 1, wherein the method comprises introducing the peptide or variant or fragment thereof into the brain of a wild-type non-human animal.
3. A method according to claim 1, wherein administration of the peptide or variant or fragment thereof to the non-human animal causes an increase in Tau protein in one or more sites in the animal's brain selected from a group consisting of: the cortex; subcortex; hippocampus; cerebellum; basal forebrain; and pons/medulla region, optionally wherein administration of the peptide or variant or fragment thereof causes an increase in Tau protein in at least one, two, three, four, five or all six sites in the animal's brain selected from a group consisting of: the cortex; subcortex; hippocampus; cerebellum; basal forebrain; and pons/medulla region.
4. A method according to claim 1, wherein administration of the peptide, or variant or fragment thereof causes an increase in Tau protein in the one or more sites in the animal's brain by at least 1%, 3%, 5%, 10% or 20% compared to an untreated control.
5. A method according to claim 1, wherein administration of the peptide, or variant or fragment thereof causes an increase in Tau protein in the one or more sites in the animal's brain by at least 30%, 40% or 50% compared to an untreated control.
6. A method according to claim 1, wherein administration of the peptide or variant or fragment thereof to the non-human animal causes a decrease in neurons in one or more sites in the animal's brain selected from a group consisting of: the cortex; subcortex; hippocampus; cerebellum; basal forebrain; and pons/medulla region, optionally wherein administration of the peptide or variant or fragment thereof causes an increase in Tau protein in at least one, two, three, four, five or all six sites in the animal's brain selected from a group consisting of: the cortex; subcortex; hippocampus; cerebellum; basal forebrain; and pons/medulla region.
7. A method according to claim 1, wherein the peptide or variant or fragment thereof, comprises or consists of at least 15, 16, 17, 18 or 19 amino acids of the sequence represented as SEQ ID NO: 3, or wherein the variant or fragment of the peptide administered to the brain of the non-human animal comprises or consists of at least 20, 21, 22, 23 or 24 amino acids of the sequence represented as SEQ ID NO: 3.
8. A method according to claim 1, wherein the peptide or variant or fragment thereof comprises or consists of at least 25, 26, 27, 28 or 29 amino acids of the sequence represented as SEQ ID NO: 3.
9. A method according to claim 1, wherein the peptide or variant or fragment thereof comprises or consists of at least 15, 20, 25 or 30 amino acid residues and has at least 90% or 95% sequence identity with SEQ ID No: 3.
10. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof being administered to the animal is less than 1 mM, or less than 750 μM, or less than 500 μM, or less than 400 μM, or less than 300 μM, or less than 200 μM, or less than 100 μM, or less than 75 μM, or less than 60 μM.
11. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof is less than 50 μM, or less than 40 μM, or less than 30 μM, or less than 20 μM, or less than 10 μM, or less than 5 μM, or less than 3 μM.
12. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof being administered is more than 0.01 μM, or more than 0.1 μM, or more than 1 μM, or more than 3 μM, or more than 5 μM, or more than 10 μM, or more than 20 μM.
13. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof being administered is more than 30 μM, or more than 40 μM, or more than 50 μM, or more than 60 μM, or more than 70 μM, or more than 80 μM, or more than 90 μM.
14. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof being administered is between 0.01 μM and 1000 μM, or between 0.1 μM and 500 μM, or between 1 μM and 100 μM, or between 1 μM and 90 μM.
15. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof is between 0.1 μM and 80 μM, or between 0.1 μM and 70 μM, or between 0.1 μM and 60 μM, or between 0.1 μM and 50 μM, or between 0.1 μM and 40 μM, or between 0.1 μM and 30 μM, or between 0.1 μM and 20 μM, or between 0.1 μM and 10 μM.
16. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof is between 10 μM and 80 μM, or between 20 μM and 80 μM, or between 30 μM and 70 μM, or between 40 μM and 60 μM.
17. A method according to claim 1, wherein the concentration of the peptide or variant or fragment thereof is 0.1-99 μM.
18. A method according to claim 1, wherein the peptide or variant or fragment thereof is introduced into the basal forebrain region of the brain.
19. A method according to claim 1, wherein the peptide or variant or fragment thereof is introduced into: (i) the medial septum/diagonal band of Broca (SID13) region of the brain; (ii) the cortical cholinergic system; and/or (iii) the nucleus basalis magnocellularis (NBM).
20. A method according to claim 1, wherein the non-human animal is a mammal.
21. A method according to claim 1, wherein the animal is a primate, optionally a monkey.
22. A method according to claim 1, wherein the non-human animal is a rodent, optionally a mouse or a rat.
23. A method according to claim 1, wherein the peptide or a variant or fragment thereof contributes to, or causes, neurodegeneration.
24. A method according to claim 1, wherein the peptide or variant or fragment thereof administered to the animal model causes cellular degeneration and thereby impairment of a testable brain function, wherein impairment of the same brain function in a human is indicative of a neurological disorder.
25. A method according to claim 1, wherein the method or model is used to investigate any neurodegenerative disease characterised by tauopathy.
26. A method according to claim 1, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); Lewy-body dementia; and Frontotemporal Dementia.
27. A method according to claim 1, wherein neurodegenerative disease is Alzheimer's Disease, Parkinson's Disease or Motor Neuron Disease.
28. A method according to claim 24, wherein the testable brain function, the impairment of which is tested, is cognitive function or attentional deficit.
29. A method according to claim 1, wherein the method comprises testing the animal model for the impairment of an appropriate brain function, optionally by providing the animal with an attentional task to test an attentional impairment.
30. A method according to claim 1, wherein the method further comprises administering prior to, simultaneously or after the peptide, or variant or fragment thereof, a test agent and determining whether the agent inhibits, prevents or increases impairment of a testable brain function and/or inhibits, prevents or increases cellular damage in the brain.
31. A method according to claim 1, wherein cellular damage comprises neurodegeneration, optionally wherein the damage is monitored or assessed by measuring one or more of: (i) the inhibition of activity in neuronal populations (i.e. assemblies); (ii) calcium levels; (iii) acetylcholinesterase activity levels; (iv) expression of alpha-7 nicotinic receptors in cell membranes; and (v) cell density and/or loss or reduction of NeuN-expressing cells (related to neuronal death) in specific areas.
32. An animal model for a neurodegenerative disease, which is a non-human animal treated with a peptide comprising or consisting of the amino acid sequence represented as SEQ ID NO: 3, or an active variant of fragment thereof.
33. An animal model prepared using the method according to claim 1.
34. Use of the animal model according to claim 32 to: (i) examine neurodegenerative or neuroregenerative processes; (ii) test pharmacological compounds which may modulate neurodegenerative or neuroregenerative processes; or (iii) screen neurodegenerating or neuroregenerating drugs.
35. A method of identifying a candidate agent, for use in the treatment, prevention or amelioration of neurodegenerative disorder, the method comprising: administering a candidate agent to the animal model according to claim 32; and determining if the candidate agent inhibits, prevents or increases impairment of a testable brain function and/or causes improvement or deterioration of cellular damage in the brain, wherein inhibition or prevention of impairment of the testable brain function, or improvement of cellular damage in the brain indicates that the test agent is a candidate for the treatment, prevention or amelioration of neurodegenerative disorder, whereas increasing impairment of the testable brain function or deteriorating cellular damage in the brain indicates that the agent is not a candidate for the treatment, prevention or amelioration of neurodegenerative disorder.
36. A method according claim 35, wherein the testable brain function is a cognitive function or an attentional deficit, optionally wherein the method comprises testing the animal model for impairment or a cognitive function or an attentional deficit.
37. A method of testing a test agent for biological activity in a neurodegenerative disease, wherein the method comprises administering the test agent to the animal model according to claim 32, and assessing the animal having a brain lesion for any change, either improvement or deterioration, associated with the brain lesion.
38. A method according to claim 37, wherein the assessment comprises determining whether the agent inhibits, prevents or increases impairment of an appropriate testable brain function, optionally a cognitive function such as attention or memory, and/or determining whether there is any improvement or deterioration in cellular damage at the relevant site(s) in the brain.
39. A method according to claim 37, wherein the test agent is a drug compound.
Description
[0081] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: —
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EXAMPLES
[0097] Despite the increasing numbers of studies targeting the primary events in neurodegeneration, there is no animal model which closely reproduces the full pathological profile (e.g. of Alzheimer's disease), since the basic mechanisms of neurodegeneration are still poorly understood. Thus, the inventors have developed a novel in vivo animal model to elucidate the basic mechanisms inducing neurodegeneration, and, importantly, in which novel test agents could be tested to determine neuroprotective (or neurotoxic) activity.
[0098] The invention involves the use of a peptide cleaved from the C-terminus of acetylcholinesterase (AChE), T30 (SEQ ID NO:3), which is composed by a bioactive portion, T14 (SEQ ID No: 4), and an inert fragment, T15 (SEQ ID No: 5) that interacts with the α7 nicotinic acetylcholine receptor (α7-nAChR). The inventors have previously shown that the application of AChE-derived peptide on cell lines promotes an AD-like phenotype. These effects are blocked by a novel candidate modulator of the α7-nAChR, NBP-14, which is the cyclized form of T14, and so has the sequence of cyclic SEQ ID No:4. As described below, the inventors have applied T30 or NBP14 on ex vivo brain slices and investigated their activity in modulating the endogenous T14 expression, and whether they contribute or prevent a neurodegeneration pattern.
[0099] The inventors show that the apparatus and model can be used to study neurodegeneration in a more physiological scenario, i.e. ex vivo brain slices, on α7-nAChR, p-Tau and Aβ expression, though it will be appreciated that there any many other proteins that can be measured to monitor degree and progression of neurodegenerative disorders. The model harnesses the inventors' new hypothesis which they believe accounts for the aberrant processes characterizing AD, based on the interaction between the α7 nicotinic acetylcholine receptor (α7-nAChR) and the toxic peptide, cleaved from the acetylcholinesterase (AChE) C-terminus, i.e. T30. The apparatus and models can be used to examine many molecular processes, test pharmacological compounds which may regulate these processes and provide a reliable tool for drug screening, reducing whole animal experiments.
[0100] Materials and Methods
[0101] Brain Extraction and Dissection
[0102] Following a lethal injection of anaesthesia (pentobarbital), fresh brains were extracted and the cortex, hippocampus, cerebellum and subcortical areas were dissected and immediately snap frozen in liquid nitrogen. Brains were stored at −80° C. to preserve the proteins. Due to death of one rat prior to the experiment beginning, the groups were as follows: [0103] PBS (control): n=6 [0104] 1 μM T30: n=5 [0105] 50 μM T30: n=6 [0106] 100 μM T30: n=6
[0107] Brain Homogenisation
[0108] Brain sections were defrosted on ice, and ice cold Lysis Buffer (PBS+protease and phosphatase inhibitors at 1:100 each) was added to each brain section. Using a pestle, the tissue was homogenised as much as possible before a sonicator probe was used on a low setting, for 5 seconds at a time, whilst being kept on ice, until the tissue was fully homogenised. Samples were incubated on ice for 20 minutes before being centrifuged at maximum speed (13,000 rpm) for 30 minutes at 4° C. The supernatant was removed and used for analysis.
[0109] β-Amyloid ELISA Commercial ELISAs for β-Amyloid 42 (Invitrogen, KMB3441) were purchased along with β-Amyloid Peptide (1-42) (Abcam, ab120959). All samples were plated at 6000 μg/mL of total protein (determined by the Pierce Protein Assay) with a positive control synthesized from wild type, whole rat brain plus β-Amyloid Peptide (1-42) at 275 ng (published concentrations found in Transgenic Animal Models of AD).
[0110] Secondary controls (no primary antibody added) and chromogen blanks were also used on every plate. A standard curve of β-Amyloid Peptide (1-42) was used ranging from 0-200 pg/mL on every plate and the protocol was followed as set out by the kit (with the exception of the peptide supplied with the kit, which was replaced by an alternative, listed above). Roughly, standards (in duplicate) and samples (in triplicate) were plated and incubated at room temperature on a plate shaker for 2 hours. All standards and samples were aspirated and the plate washed before the ‘detector’ antibody supplied with the kit was added to all wells with the exception of secondary controls and chromogen blanks. A further 1 hour incubation period at room temperature on a plate shaker followed, before the antibody was aspirated and the plate washed again. IgG HRP was added to every well (with the exception of the chromogen blanks) and the plate incubated for a further 30 minutes at room temperature on a plate shaker. All solution was aspirated and the plate washed before adding Stabilized Chromogen to every well and incubating the plate for 30 minutes in the dark, at room temperature on a plate shaker. Finally, Stop Solution was added to every well and the absorbance read at 450 nm.
[0111] Total Tau ELISA
[0112] Commercial ELISAs for Total Tau detection (Abcam, ab210972) were purchased. All samples were plated at 0.5 μg/mL of total protein (determined by the Pierce Protein Assay) along with a full standard curve of Tau ranging from 0-2000 pg/mL and Secondary controls (no primary antibody added). The protocol was followed as set out by the kit, roughly, standards (in duplicate) and samples (in triplicate) were plated, followed immediately by the addition of the Antibody Cocktail (minus the Capture Antibody for the Secondary Controls) and incubated at room temperature on a plate shaker for 1 hour. All solution was aspirated and the plate washed before TMB Substrate was added to all wells and incubated for 10 minutes in the dark, at room temperature on a plate shaker. Finally, Stop Solution was added to all wells and the plate incubated for 1 minute at room temperature on a plate shaker before the absorbance was read at 450 nm.
[0113] T14 ELISA
[0114] The inventors have developed an in-house ELISA for the detection of T14. All samples left (PBS: cortex n=6, subcortex n=6, hippocampus n=0, cerebellum n=4; 1 μM T30: cortex n=5, subcortex n=3, hippocampus n=1, cerebellum n=4; 50 μM T30: cortex n=5, subcortex n=4, hippocampus n=1, cerebellum n=4; 100 μM T30: cortex n=6, subcortex n=6, hippocampus n=0, cerebellum n=6) were diluted to 1:10 and plated (in triplicate) with a full T14 standard curve (plated in duplicate) ranging from 0-40 nM and Secondary Controls. Plates were incubated overnight at 4° C. on a plate shaker and then fully aspirated before addition of BSA Blocking Solution and a further incubation of 6 hours at room temperature on a plate shaker. Blocking Solution was aspirated and primary antibody (T14 Polyclonal, Genosphere) added to all wells (with the exception of Secondary Controls) before incubating overnight at 4° C. on a plate shaker. Antibody solution was aspirated and the plate washed followed by the addition of secondary antibody and incubation for 2 hours at room temperature on a plate shaker. All solution was aspirated and the plate washed, then TMB substrate was added and the plate incubated for 15 minutes at room temperature on a plate shaker. Stopping Solution was added and the absorbance read at 450 nm.
[0115] Tissue Preparation and Immunohistochemistry
[0116] Rat brain samples were removed from PBS and cryoprotected by incubating in 30% sucrose solutions for 72 h or until saturated. Whole brains were cut in half along the midline and each half was embedded in TissueTek and stored at −80° C. until the time of cyro-sectioning.
[0117] Sagittal sections of 25 μm were cut using a cryostat starting at the midline. Sections were collected in 24-well plates, and directly used for staining or stored in a cryoprotection solution (25 mM Na-phosphate buffer pH 7.4, 30% ethylene glycol, 20% glycerol) at −20° C. until time of use. All staining were performed with sections mounted on superfrost slides.
[0118] Immunostaining for the detection of beta amyloid (Aβ), phosphorylated Tau (pTau), neurons (NeuN) and microglia (Iba1) was performed in the following manner. Sections were pretreated for antigen retrieval either in citric Buffer pH 6.0 for 30 minutes at 90° C. for pTau or with 70% formic acid for 10 min for Aβ. After antigen retrieval sections were permeabilized in 0.3% Triton X-100/PBS, blocked in 10% normal goat serum/PBS and incubated with the primary antibody diluted in 1% normal goat serum, 0.1% Triton X-100 in PBS at 4° C. overnight.
[0119] The following primary antibodies were used for immunostaining: anti-beta amyloid (Aβ) monoclonal mouse, 6E10, (1:1000; Covance, cat #39320), monoclonal mouse anti-phosphorylated Tau, AT180, (1:500; Thermo, cat #MN1040), monoclonal rabbit anti-Iba1 (1:500; Synaptic System, cat #234004), polyclonal rabbit anti-NeuN (1:500; Millipore, cat #ABN78).
[0120] Co-stainings were performed with 6E10 combined with Iba1 and AT180 combined with NeuN. Sections were washed three times in PBS for 15 min and incubated in appropriate secondary antibody (Sigma) for 2 hours at room temperature. Sections were again washed in PBS three times in PBS for 15 min, then incubated with DAPI staining to detect nuclei. Finally, mounting media was applied to stained sections and slides were coverslipped for imaging with the Zeiss AxioScan.Z1 system (Carl Zeiss Microscopy).
[0121] Image Acquisition and Quantitative Analysis
[0122] Automated image acquisition was conducted using a Zeiss AxioScan.Z1 slide scanning device (Leica Biosystems) equipped with an LED-Colibri7 light source and an Axiocam 506 mono camera set. Images were taken with 20× magnification (pixel size: 0.22 μm) in a none-confocal manner and images were visualized using Zen software. Image data was imported into the Visiopharm® image analysis software (Visiopharm A/S) to perform region selection.
[0123] Manual segmentation of the cortex, hippocampus, midbrain, basal forebrain and pons/medulla regions was performed by subdividing the images of the sagittal brain sections using coordinates published by the Allen Developing Mouse Brain Atlas (Allen Institute) as guidelines.
[0124] Image analysis scripts for characterization and quantification of intracellular and extracellular Aβ, pTau, NeuN and Iba1 were developed using Acapella® Studio 5.1 (PerkinElmer Inc.) and the integrated Acapella® batch analysis as part of the Columbus® system. For all analyses individual cells within tissue sections were identified using the DAPI signal and a customised nuclei detection workflow based on the Acapella® “nuclei_detection_B” algorithm. Several quality control parameters were implemented to discard out out-of-focus nuclei and non-nuclear structures. These include e.g. applying thresholds for minimum signal contrast, nuclear area and nuclear roundness. Cytoplasm of detected cells was defined as a 4-pixel-wide concentric ring around the previously segmented nuclei (perinuclear area). Outside this perinuclear ring, a 3-pixel-wide background area was created, serving as cellular individual and, after median aggregation, whole-brain-region reference region for determination of NeuN- and Iba1-positive cellular populations.
[0125] Signal intensities for Aβ, pTau, NeuN and Iba1 stainings were evaluated in all cellular sub regions. Cells were identified as being NeuN- or Iba1-positive when the average signal intensity in the nuclear area was at least 1.5 or 2 times higher than the brain region median background, respectively.
[0126] Extracellular plaques were segmented by applying an intensity threshold to the image: signal with at least 2 times higher intensity than the median cellular amyloid background was considered potentially belonging to a plaque. To exclude false-positive plaques from analysis, further filtering of these initial plaque-like objects was achieved by applying thresholds for minimum plaques size (i.e. >200 p×2) and axial ratio (length small axis/length long axis >0.4). All readouts were calculated as average values per brain region and histological section. These values were then used to calculate respective averages per animal.
[0127] Data Handling and Analysis
[0128] A total of 16 animals were used for the study, with N=8 animals per treatment group.
[0129] Quantitative results for six sections per animal were averaged to generate one data point per animal. Statistical analysis was performed using an unpaired t test. *p<0.05; **p<0.01 T30 peptide vs. saline.
[0130] Antibodies Used for Immunohistological Analysis of Brain Samples of Sprague-Dawley Rats.
TABLE-US-00006 AD related pathology Phenotype detected Primary antibody Aβ Plaque Beta amyloid 6E10 Tau Phosphorylated tau AT8 Gliosis Activated microglia Iba1 Cell loss Neronal cell count NeuN
[0131] Analysis
[0132] Initially, the standard deviation of the blanks, Limit of Detection (LOD) (standard deviation of the Blanks×3.3) and Limit of Quantification (LOQ) (standard deviation of the Blanks×10) were calculated from the absorbance values (A.sub.450). If applicable, the average of the Chromogen Blanks was taken away from all standard curve, sample and control values, followed by the average of the Blanks and then the average of the Secondary Controls. All values above the LOQ were used to plot graphs and interpolate values (if applicable) as pg/mL using GraphPad Prism Software. All statistical analysis was performed using GraphPad Prism Software.
[0133] Human Tau SimpleStep ELISA Kit—Abeam ab210972:
[0134] The protocol is as follows: [0135] Prepare all reagents, working standard, and samples. [0136] Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal and return to 4° C. storage. [0137] Add 50 μl of all sample or standard to appropriate wells. [0138] Add 50 μl of the antibody cocktail to each well. [0139] Seal the plate and incubate for 1 hour at room temperature on a plate shakers set to 400 rpm. [0140] Wash each well with 3×350 μl 1× wash buffer PT. Wash by aspirating or decanting from wells and then dispensing 350 μl 1× wash buffer PT into each well. Complete removal of liquid at each step is important for good performance. After the last wash, invert the plate and blot it against clean paper towels to remove excess liquid. [0141] Add 100 μl of TMB substrate to each well and incubate for 10 minutes in the dark on a plate shaker set to 400 rpm. [0142] Add 100 μl of stop solution to each well. Shake plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm, and this is an
[0143] In addition, secondary controls were added to all plates which were subtracted from all A.sub.450 Values during the normalisation. [0144] Peptide for the standard Curve, dilution of the standards and all samples were diluted in 1× Cell Extraction Buffer (5× Cell Extraction Buffer PTR provided with the kit) plus 1× Cell Extraction Enhancer Solution (50× Cell Extraction Enhancer Solution provided with the kit) in dH.sub.2O. [0145] 1× Wash Buffer prepared by diluting 10× Wash Buffer PT (provided with the kit) with dH.sub.2O. [0146] Antibody Cocktail: [0147] 1× Human Tau Capture Antibody+1× Human Tau Detector Antibody (both provided with kit in 10× form) diluted in Antibody Diluent CPI (provided with the kit). [0148] Antibody for Secondary Controls: [0149] 1× Human Tau Detector Antibody (provided with kit in 10× form) diluted in Antibody Diluent CPI (provided with the kit).
[0150] Statistical Analysis: [0151] Average A.sub.450 of the Blanks was subtracted from all Standard and Sample A.sub.450 Values [0152] Average of A.sub.450 of the Secondary Controls was subtracted from all Standard and Sample A.sub.450 Values [0153] An Ordinary One Way ANOVA was performed on each brain area against the Control for that area, with Dunnett's Multiple Post-Hoc Comparisons test.
[0154] Morris Water Maze Method
[0155] The 2.1 m diameter black water maze pool is filled to a depth of 40 cm with 22 degree C. water. This leaves the 15-cm diameter submerged platform 1 cm below the water level. The rat is then placed in the water at one of the cardinal points (N, E, S, W) quadrant and allowed 2 minutes to find the platform. If the rat finds the platform within this time it is allowed 15 seconds on the platform before it is removed, gently towelled down and placed under a warming lamp. If the rat does not find the platform within the 2 minutes it is led to the platform by trailing a hand in the water in front of the rat, leading it to the platform. It is then allowed 15 seconds on the platform before it is removed, towelled down and placed under a warming lamp. The routine is repeated 4 times per day (maximum 10 days, although the current quote allows for 6 days of testing with 4 days of reversal learning) until the rat has clearly learnt the maze, signified by no significant improvement occurring after 3 consecutive days. The inter-trial interval time between swims is 10 minutes, A probe trial is run at the end of both reference memory trials and reversal learning trials to probe working memory.
Example 1
[0156] The primary objective was to establish whether a single dose of T30, injected into the basal forebrain of WT rats, could induce, neurochemically, an ‘Alzheimer's-like’ profile, defined as statistically significant increases in AD-related proteins in treatment groups compared to control. Secondly, the objective of this work was to establish at which concentration T30 caused these changes.
[0157] Stereotaxic injection of either PBS (control), or one of 3 doses of T30 (1 M, 50 μM and 100 μM) into MS/VDB (Medial Septum/Vertical Limb of the Diagonal Band) of adult male Lister hooded rats was performed at Nottingham University. Rats were culled 2-3 weeks after injection and brains were extracted and dissected to separate cortex, hippocampus, cerebellum and subcortical areas for neurochemical analysis at Neuro-Bio. Each brain area was analysed for levels of total Tau, β-Amyloid 42 and T14.
[0158] Results
Example 1—β-Amyloid (42)
[0159] Referring to
[0160] Note all data for β-Amyloid 42 is shown normalised to Positive Control instead of in pg/mL. Due to the difficulties with the assay, it was decided that pg/mL would give an inaccurate quantification and therefore an unreliable representation of the data.
Example 2—Total Tau
[0161] Referring to
[0162] Referring to
Example 3—T14
[0165] Referring to
Example 4—Effects of AChE-Derived Peptide (T30) on Alzheimer's Disease Related Parameters (pTau, NeuN) in Sprague-Dawley Rats
[0166] Sagittal brain sections from Sprague-Dawley rats receiving either, an acute administration of T30 peptide or saline, were prepared using a cryostat as described in the methods. Every sixth section was collected starting at the midline and six sections per animal were immunostained for detection of AR (6E10), pTau (pS202/pT205), microglia (Iba1) and neurons (NeuN). Primary antibodies were combined in two co-staining sets for all animal samples. Quantitative analysis for the different markers was performed in 5 different regions of interest (ROIs) and include, cortex, hippocampus, basal forebrain, midbrain and pons/medulla.
[0167] Referring to
[0168] Referring to
Example 5—Effects of AChE-Derived Peptide (T30) on Alzheimer's Disease Related Parameters (6E10, Iba1) in Sprague-Dawley Rats
[0169] Sagittal brain sections from Sprague-Dawley were prepared and IHC was performed in the second set of co-staining for detection of Aβ and Iba1. No specific intracellular or extracellular Aβ immunoreactivity was observed in the hippocampus, cortex, midbrain, basal forebrain or pons/medulla of saline or T30 peptide treated rats (
Example 6—Animal Model Behavioural Study
[0170] 1) Morris Water Maze Timepoint 1
[0171] Both the MWM 6-day learning curve and the further 4-day reversal learning curve indicate that there are no significant differences in treatment groups at any day. Two-way ANOVA with repeated measures (Genotype X Day). The Probe Trial (PT) and Reversal Probe Trial (RPT) there were no significant differences between the treatment groups for their time spent in, or visits to the Target Quadrant. Two-way ANOVA (Genotype X Quadrant).
[0172] Referring to
[0173] While the probe trial revealed good discrimination for the target quadrant in both treatment groups; this was less prominent in the peptide group during the reversal probe trial for visits into the target quadrant and target platform zones. This was indicated by there being no significant difference between visits to the target platform and quadrant zones and the zones previously a target in the probe trial.
[0174] 2) Morris Water Maze Timepoint 2
[0175] Both the MWM 4-day learning curve and the further 4-day reversal learning curve indicate that there are no significant differences in treatment groups at any day. Two-way ANOVA with repeated measures (Genotype X Day). In the Probe Trial (PT) and Reversal Probe Trial (RPT) there were no significant differences between the treatment groups for their time spent in, or visits to the Target Quadrant. Two-way ANOVA (Genotype X Quadrant).
[0176] Referring to
[0177] 3) Morris Water Maze Timepoint 3
[0178] Referring to
Example 6—Administration of T20
[0179] Referring to
CONCLUSIONS
[0180] Total Tau
[0181] Total Tau levels were surprisingly found to be significantly increased in all brain regions (cortex, subcortex, hippocampus and cerebellum) for the intermediate doses (1 μM and 50 μM) of the T30 peptide, with levels returning to that of controls for the highest dose (100 μM). In all regions, the 1 μM T30 dose showed the greatest increase in Total Tau levels.
[0182] β-Amyloid 42
[0183] No significant differences were found in the levels of β-Amyloid in any region of the dissected brains (cortex, subcortex, hippocampus or cerebellum) following a single injection of T30 peptide into the basal forebrain, 2-3 weeks before rats were sacrificed. Previous research (Lin et al, 2009, J. Alzheimer's Dis, 18(4):907-18) has clearly established that increased total Tau but not β-amyloid in CSF correlates with short-term memory impairment in Alzheimer's disease. The results described herein are not inconsistent with these earlier findings of unaltered β-Amyloid levels despite significantly elevated Tau levels.
[0184] T14
[0185] There were no significant differences in the levels of T14 at any concentration of T30 in any of the samples analysed (cortex, subcortex and cerebellum) compared to controls. There were no hippocampal samples remaining to be analysed for T14 levels and there were limited numbers of other regions.
[0186] NeuN Positive Cells
[0187] The density of NeuN positive or expressing cells was significantly decreased in the midbrain, while no differences were observed in the other brain regions (cortex, hippocampus, basal forebrain or pons/medulla). NeuN levels are indicative of the number of mature neurons present.
[0188] Summary
[0189] As shown in the Figures, T30 peptide treatment induces a highly significant, dose-dependent increase in Tau in all four brain areas studied. In all cases, the highest dose (i.e. 100 M) was no different from the PBS-injected controls, which the inventors hypothesise is most likely due to a shutting down of the calcium channel when excessively stimulated (Standen, 1981, “Ca inactivation by intracellular Ca injection into Helix neurons”, Nature 293, 158-159) as seen previously with high doses of peptide applied to breast cancer cell cells (Onganer et al., 2006, “An acetylcholinesterase-derived peptide inhibits endocytic membrane activity in a human metastatic breast cancer cell line”, Biochimica et Biophysica Acta, 1760(3):415-420]) and alpha 7 transfected oocytes (Greenfield et al., 2004, “A novel peptide modulates alpha 7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain”. J Neurochem 90, 325-331) as well as in brain slices (Bon et al., 2003, “Bioactivity of a peptide derived from acetylcholinesterase: electrophysiological characterization in guinea-pig hippocampus”. Eur J Neurosci 17, 1991-1995) and organotypic hippocampal neurons (Day and Greenfield 2004, “A non-cholinergic, trophic action of acetylcholinesterase on hippocampal neurones in vitro”: Molecular mechanisms. Neuroscience 111, 649-656).
[0190] However, in lower doses (less than 100 μM), where the enhanced calcium influx is viable, the T30 peptide induces activation of GSK (Garcia-Rates et al., 2016, “(I) Pharmacological profiling of a novel modulator of the α7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains”. Neuropharmacology, vol 105, pp. 487-499) leading in turn to increased phosphorylation of Tau (Rankin et al., 2007, “Tau phosphorylation by GSK-3β promotes tangle-like filament morphology”. Mol Neurodegener 2: 12), in turn promoting the formation of tangles, the cardinal marker of AD (Braak and Braak 2011, “Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years”. J Neuropathol Exp Neurol. 70(11):960-9). In other words, the inventors have surprisingly shown that low μM doses of T30 are receptor-mediated, whereas high doses are not receptor-mediated, and this was totally unexpected. The inventors believe, therefore, that the dose range of 1-99 μM T30 at which it is receptor-mediated is optimum and preferred.
[0191]
[0192] (1) T30 binds to the allosteric site of the receptor to enhance the opening of the channel for Ca.sup.2+ influx into the cell (Greenfield et al., 2004, “A novel peptide modulates alpha 7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain”. J Neurochem 90, 325-331
[0193] (2) Calcium entry induces depolarization and opening of the voltage-dependent (L-VOCC) channel allowing still more Ca.sup.2+ into the cell (Dickinson et al., 2007, “Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells”. J. Neurochem. 2007 February; 100(4):1089-96);
[0194] (3) This raised intracellular calcium induces an increase in AChE G4 release that includes T30 (Greenfield, 2013, “Discovering and targeting the basic mechanism of neurodegeneration: the role of peptides from the c-terminus of acetylcholinesterase Chemico-Biological Interactions”. 203(3):543-6);
[0195] (4) Calcium also induces upregulation of the α7 nicotinic receptor that will allow more Ca.sup.2+ get in the cell by providing still more targets for T30 (Bond et al., 2009, “Upregulation of alpha 7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides”. Plos One, 4);
[0196] (5) Calcium activates enzymes (i.e. GSK-3) that will (a) increase Tau, (b) activate y-secretase/β-secretase that will trigger cleavage of extracellular toxic Amyloid that (c) together with T30 will promote a still further toxic amount of Ca.sup.2+ into the cell. (Hartigan & Johnson (1999, “Transient increases in intracellular calcium result in prolonged site-selective increases in Tau phosphorylation through a glycogen synthase kinase 3beta-dependent pathway”. J Biol Chem. 23; 274(30):21395-401), Cai et al. (2012, “Roles of glycogen synthase kinase 3 in Alzheimer's disease”. Curr Alzheimer Res. 9(7):864-79.), Garcia-Rates et al (2013, “Additive Toxicity of β-Amyloid by a Novel Bioactive Peptide In Vitro: Possible Implications for Alzheimer's Disease”. PLoS ONE 8(2):e54864.)).