Animal models for neurodegenerative disorders

Abstract

This invention relates to animal models, and more particularly to novel in vivo animal models for neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, or motor neuron disease. The invention extends to methods for providing such models. The invention also provides the animal models themselves and methods for studying the underlying mechanisms present in such neurodegenerative disorders, particularly Alzheimer's disease, and further extends to models, methods, and assays for testing pharmacological test compounds and screening drugs for the treatment of neurodegenerative diseases, said pharmacological test compounds being capable of modulating neural processes.

Description

[0001] This invention relates to animal models, and more particularly to novel in vivo animal models for neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, or motor neuron disease, and to methods for providing such models. The invention provides the animal models themselves and methods for studying the underlying mechanisms present in such neurodegenerative disorders, particularly Alzheimer's disease, and extends to models, methods, and assays for testing pharmacologically indicated compounds and for drug screening for the treatment of neurodegenerative diseases, wherein the pharmacologically indicated compounds can modulate neurological processes.

[0002] Alzheimer's disease (AD) is the most common form of dementia, but the major events contributing to this disorder remain unsolved. The most popular "amyloid hypothesis" is now increasingly challenged, thus necessitating alternative theories compatible with all clinical features. One such approach focuses on the distinctive characteristics of neurons in AD that are selectively and primarily vulnerable. These neurons form a continuous central structure of adjacent cell populations extending from the basal forebrain (BF) into the midbrain and brainstem, projecting onto several brain regions such as the cortex, hippocampus, and olfactory bulb. Despite the heterogeneity of neurotransmitters within this core of susceptible cells, a common and interesting feature is that they contain an enzyme, acetylcholinesterase, which has now been identified as performing a non-cholinergic function. This non-enzymatic action regulates calcium ion inflow into neurons; therefore, it can be either nutrient-rich or toxic, depending on dosage, availability, and neuronal age.

[0003] Acetylcholinesterase (AChE) is expressed in various forms at different stages of development, all with the same enzymatic activity, but each form has a very different molecular composition. "T-AChE" is expressed at the synapse, and the inventors had previously identified two peptides that can be cleaved from the C-terminus: one called "T14" (14-terminal peptide), located within the other, and the other called "T30" (30-terminal peptide), both showing high sequence homology to comparable regions of β-amyloid protein. The AChE C-terminal peptide "T14" has been identified as the silencing part of the AChE molecule, responsible for its range of non-hydrolytic activity. The synthesized 14-amino acid peptide analog (i.e., "T14") and subsequently the larger, more stable, and more efficient amino acid sequence into which it was inserted (i.e., "T30") exhibited similar activity to the reported "non-cholinergic" AChE, where the inert residues within the T30 sequence (i.e., "T15") had no effect.

[0004] Currently, there are no widely accepted in vivo animal models that can reproduce the full pathological profile of neurodegenerative disorders such as Alzheimer's disease (AD) because the underlying mechanisms of neurodegeneration remain poorly understood. Current systems not only fail to replicate the full clinical profile of the disease, but most available systems rely on transgenic animals to reflect diseases with a clear genetic basis, which occur in only a small percentage of cases. Furthermore, the production of transgenic animals is very expensive, and there is a long waiting period for the damage to become apparent. Therefore, there is an urgent need for improved animal models or assays that enable precise research into neurodegenerative diseases.

[0005] The inventors have developed a hypothesis that they believe explains the aberrant processes characterizing Alzheimer's disease, based on the interaction between the α7 nicotinic acetylcholine receptor (α7-nAChR) and a toxic 30-meric peptide, T30, which is cleaved from the C-terminus of acetylcholinesterase (AChE). Based on this hypothesis, they have established a novel, non-genetically modified approach using in vivo (i.e., rodent) models that can be used to study neurodegenerative disorders in far more physiological settings than cell cultures.

[0006] Therefore, the inventors administered a single dose of peptide T30 to the mid-septal / basal forebrain of rats and investigated T30-mediated modifications on the toxic peptide (T14) and two Alzheimer's disease markers (τ and Aβ) in four different brain regions: the cortex, subcortex, hippocampus, and cerebellum. Furthermore, they analyzed the basal forebrain and pons / medulla regions of the brain using immunohistochemistry, which employed antibody-based quantification. The overall objective was first to determine whether a single dose of T30 could neurochemically induce an "Alzheimer's-like" profile, defined as a statistically significant increase in AD-related proteins in the treatment group compared to control, and second to determine at what concentration of T30 these changes were induced. Figure 2 and 3 The ELISA results shown surprisingly revealed an increase in total τ protein levels in all four brain regions (cortex, subcortex, hippocampus, and cerebellum) upon administration of T30-peptide. τ protein is a well-known major pathological marker of Alzheimer's disease; therefore, the methodology described here clearly demonstrates the role of T30 in triggering Alzheimer's-like profiling in these four brain regions. Furthermore, as... Figure 15 As shown, compared with saline-treated animals, a significant decrease in the density of NeuN-positive (i.e., NeuN-expressing) cells was observed in the midbrain of rats administered T30 peptide; NeuN-positive cells are a marker of mature neurons. Furthermore, Figure 15 The study also demonstrated a deterioration in the behavior of rats treated with T30 using the Morris water maze test.

[0007] When considering the data holistically, the inventors firmly believe this is the first evidence that a toxin (i.e., the T30 peptide) triggers a consistent Alzheimer's-like biochemical profile in the brains of other normal wild-type rodents. The method described herein proposes a highly novel in vivo approach for monitoring and manipulating neurochemical phenomena contributing to neurodegeneration in a time-dependent and site-specific manner. This novel approach clearly allows for the exploration of early stages occurring during neurodegeneration in a physiological context, maintaining local neuronal circuits in the study area, and providing the possibility of monitoring their acute responses. This methodology can be applied to examine numerous molecular processes, test pharmacological compounds, and provide a reliable tool for drug screening that can modulate these processes.

[0008] Therefore, in a first aspect of the invention, a method for providing an animal model for a neurodegenerative disease is provided, the method comprising introducing a peptide into the brain of a non-human animal, the peptide comprising an active variant of, or consisting of, an amino acid sequence represented as SEQ ID NO: 3 or a fragment thereof, wherein the peptide causes an increase in τ protein at one or more sites in the brain of the animal.

[0009] Preferably, the method comprises introducing a peptide or a variant or fragment thereof into the brain of a wild-type non-human animal. Advantageously, the inventors surprisingly observed that, following the administration of the toxic T30 peptide into the brain of a wild-type (i.e., in other words, normal) non-human animal, the levels of total τ protein increased in the cortex, subcortex, hippocampus, and cerebellum of the animal. Interestingly, the inventors did not observe any significant differences in β-amyloid protein levels in any region of the dissected brain after administration of T30. However, previous studies (Lin et al., 2009, J. Alzheimer's Dis, 18(4): 907-18) have established that increased total τ protein, but not β-amyloid protein, in the CSF is associated with short-term memory impairment in Alzheimer's disease, and therefore, the results described herein are inconsistent with these earlier findings. Therefore, advantageously, the method of the present invention preferably leads to the development of new animal models of τ protein lesions, which represent neurodegenerative disorders or neurological disorders.

[0010] Therefore, in a second aspect of the invention, an animal model for neurodegenerative diseases is provided, the animal model being a non-human animal treated with a peptide comprising, or consisting of, an active variant of, an amino acid sequence represented as SEQ ID NO: 3 or a fragment thereof.

[0011] Figure 3The administration of T30 peptide was shown to surprisingly lead to: (i) an increase of approximately 40%–50% in tau protein in the cortex of animal models; (ii) an increase of approximately 175%–200% in tau protein in the subcortex; (iii) an increase of approximately 30%–60% in tau protein in the hippocampus; and (iv) an increase of approximately 160%–180% in the cerebellum. The inventors were surprised to achieve such high levels of tau protein with such low levels of T30 peptide (i.e., 1 μM or 50 μM). Furthermore, Figure 8 The administration of the T30 peptide was shown to surprisingly reduce the density of NeuN-positive cells (i.e., those associated with mature neurons) in the midbrain of treated animals. Similar to the τ protein, the inventors were surprised to achieve such a low number of NeuN cells or neurons when such low levels of the T30 peptide (i.e., 1 μM or 50 μM) were administered.

[0012] Therefore, preferably, the peptide is introduced into the brain of a non-human animal (preferably a normal wild-type animal) comprising, or consisting of, an active variant of, the amino acid sequence represented as SEQ ID NO: 3 or a fragment thereof, to create an animal model of the second aspect showing an increase in the τ protein at one or more sites in the animal's brain. Furthermore, preferably, the peptide is introduced into the brain of a non-human animal (preferably a normal wild-type animal) comprising, or consisting of, an amino acid sequence represented as SEQ ID NO: 3 or an active variant of, the fragment thereof, to create an animal model of the second aspect showing a reduction in neurons at one or more sites in the animal's brain.

[0013] Preferably, in the method of the first aspect or the model of the second aspect, administration of the peptide or its variants or fragments to a non-human animal causes an increase in τ protein or a decrease in neurons at one or more sites in the animal's brain, these sites being selected from the group consisting of: the cortex; the subcortex; the hippocampus; the cerebellum; the basal forebrain; and the pons / medulla region. Preferably, administration of the peptide or its variants or fragments causes an increase in τ protein or a decrease in neurons at at least two, three, four, five, or all six sites in the animal's brain, these sites being selected from the group consisting of: the cortex; the subcortex; the hippocampus; the cerebellum; the basal forebrain; and the pons / medulla region.

[0014] Preferably, compared with an untreated control, the administration of the peptide or its variants or fragments causes a statistically significant increase in τ protein at one or more sites in the animal's brain, preferably an increase of at least 1%, or more. Preferably, compared with an untreated control, the administration of the peptide or its variants or fragments causes an increase in τ protein at one or more sites in the animal's brain by at least 3%. Preferably, compared with an untreated control, the administration of the peptide or its variants or fragments causes an increase in τ protein at one or more sites in the animal's brain by at least 5%, 10%, or 20%. More preferably, compared with an untreated control, the administration of the peptide or its variants or fragments causes an increase in τ protein at one or more sites in the animal's brain by at least 30%, 40%, or 50%.

[0015] Preferably, compared to an untreated control, administration of the peptide or its variants or fragments causes a statistically significant reduction, preferably at least a 1% increase, or more, in neurons at one or more sites in the animal's brain (and preferably in its midbrain). Preferably, compared to an untreated control, administration of the peptide or its variants or fragments causes a reduction of at least 3% in neurons at one or more sites in the animal's brain (and preferably in its midbrain). Preferably, compared to an untreated control, administration of the peptide or its variants or fragments causes a reduction of at least 5%, 10%, or 20% in neurons at one or more sites in the animal's brain (and preferably in its midbrain). More preferably, compared to an untreated control, administration of the peptide or its variants or fragments causes a reduction of at least 30%, 40%, or 50% in neurons at one or more sites in the animal's brain (and preferably in its midbrain).

[0016] Acetylcholinesterase is a serine protease that hydrolyzes acetylcholine and is well known to those skilled in the art. The major form of acetylcholinesterase found in the brain is called tailed acetylcholinesterase (T-AChE). Furthermore, one embodiment of tailed acetylcholinesterase (Gen Bank: AAA68151.1) has a protein sequence of 614 amino acids and is provided herein as SEQ ID No: 1, as follows:

[0017] 1mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv

[0018] 61saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn

[0019] 121relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm

[0020] 181nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv

[0021] 241gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel

[0022] 301vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg

[0023] 361vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe

[0024] 421dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy

[0025] 481eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq

[0026] 541yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn

[0027] 601qfdhyskqdr csdl

[0028] [SEQ ID No:1]

[0029] The first 31 amino acid residues of SEQ ID NO: 1 were removed, and the protein was released, leaving a 583-amino acid sequence.

[0030] The inventors have compared the sequence of β-amyloid protein (Aβ) with three peptides derived from the C-terminus of AChE (hereinafter referred to as T30, T14 and T15, and described below).

[0031] The partial amino acid sequence of β-amyloid protein (Aβ) is provided herein as SEQ ID No: 2, as follows:

[0032] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

[0033] [SEQ ID No:2]

[0034] The amino acid sequence of T30 (which corresponds to the last 30 amino acid residues of SEQ ID NO: 1) is provided herein as SEQ ID NO: 3, as follows:

[0035] KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

[0036] [SEQ ID No:3]

[0037] The amino acid sequence of T14 (which corresponds to 14 amino acid residues positioned toward the end of SEQ ID NO: 1 and lacks the last 15 amino acids found in T30) is provided herein as SEQ ID NO: 4, as follows:

[0038] AEFHRWSSYMVHWK

[0039] [SEQ ID No:4]

[0040] The amino acid sequence of T15 (which corresponds to the last 15 amino acid residues of SEQ ID NO: 1) is provided herein as SEQ ID NO: 5, as follows:

[0041] NQFDHYSKQDRCSDL

[0042] [SEQ ID No:5]

[0043] The peptides used to prepare the animal models of the present invention can be derived from acetylcholinesterase itself (i.e., SEQ.ID.No.1) or its active variants or fragments, including modified forms of peptides with modified amino acid residues, such as biotinylated forms. Variants of the peptide of SEQ ID NO:3 include peptides having 1, 2, or 3 amino acid substitutions and / or 1, 2, or 3 amino acid deletions and / or 1, 2, or 3 added amino acid residues compared to SEQ ID NO:3. Suitable variants may, for example, have N-terminal and / or C-terminal extensions. Using the given SEQ ID NO:3 as a guide for comparison, it is straightforward to prepare variant peptides and test their potency in the methods and models according to the present invention. For example, one can begin by testing the same peptide as SEQ ID No:3 (except for one or two conserved substituted amino acid residues). Conserved substitutions can be predicted based on the characteristics of well-characterized amino acids. It is also possible to determine the active variants of SEQ ID NO:3 used according to the present invention by in vitro assays of peptides that retain calcium channel regulatory activity. For this purpose, for example, midbrain slices from guinea pigs can be used for electrophysiological studies as previously described in WO 97 / 35962. Alternatively, for example, organotype tissue cultures from hippocampal slices from rats can be used.

[0044] Preferably, the active variant or fragment of the peptide administered to the brain of a non-human animal comprises or consists of at least 15, 16, 17, 18, or 19 amino acids of the sequence represented by SEQ ID NO: 3. More preferably, the active variant or fragment of the peptide administered to the brain of a non-human animal comprises or consists of at least 20, 21, 22, 23, or 24 amino acids of the sequence represented by SEQ ID NO: 3. Even more preferably, the active variant or fragment of the peptide administered to the brain of a non-human animal comprises or consists of at least 25, 26, 27, 28, or 29 amino acids of the sequence represented by SEQ ID NO: 3. Preferably, the active variant or fragment of the peptide administered to the brain of a non-human animal comprises or consists of fewer than 40, 39, 38, 37, 36, or 35 amino acids of the sequence represented by SEQ ID NO: 3. Most preferably, the peptide administered to the brain of a non-human animal comprises or consists of 30 amino acids, i.e., SEQ ID NO: 3. Suitable variants of SEQ ID NO:3 used in this invention may be peptides containing at least 15 amino acid residues and having at least 70% sequence identity with part or all of the AChE sequence of SEQ ID NO:1. Preferably, the peptide used in this invention contains at least 15, 20, 25, or 30 amino acid residues and has at least 90% or 95% sequence identity with SEQ ID NO:3.

[0045] The ends of peptides or their variants or fragments may be protected by N- and / or C-terminal protecting groups having properties similar to acetyl or amide groups. Peptides or their variants or fragments may be biotinylated or tritized. Peptides may be synthetic peptides prepared by chemical synthesis, or they may be prepared from larger peptide or polypeptide molecules by enzymatic digestion, or they may be generated by recombinant technologies.

[0046] This method (or assay) involves administering an effective amount of a peptide comprising, or consisting of, the amino acid sequence represented as SEQ ID NO: 3 or its active variants or fragments, such that the peptide causes elevated levels of the τ protein in the brain. One or more doses of the peptide or its variants or fragments may be administered to an animal. Preferably, the concentration of the peptide or its variants or fragments administered to the animal may be 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. Preferably, the concentration of the peptide or its variants or fragments may be 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.

[0047] Preferably, the concentration of the applied peptide, or its variant or fragment, can be greater than 0.01 μM, or greater than 0.1 μM, or greater than 1 μM, or greater than 3 μM, or greater than 5 μM, or greater than 10 μM. Preferably, the concentration of the peptide, or its variant or fragment, can be greater than 20 μM, or greater than 30 μM, or greater than 40 μM, or greater than 50 μM. Preferably, the concentration of the peptide, or its variant or fragment, can be greater than 60 μM, or greater than 70 μM, or greater than 80 μM, or greater than 90 μM.

[0048] It will be appreciated that any of the above concentrations of peptides, their variants, or fragments can be combined in any combination. For example, the concentration of the applied peptide, or its variant, or fragment can be 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. Preferably, the concentration of the peptide, or its variant, or fragment can be 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. Preferably, the concentration of the peptide, or its variant, or fragment can be 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. Preferably, the concentration of the peptide or its variants or fragments can be 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. In the most preferred embodiment, about 1 μM or 50 μM of T30 or its variants or fragments is administered to the brain of a non-human animal. Therefore, any of the above upper and lower limits can be combined with each other.

[0049] Figure 8 This study demonstrates how administration of the T30 peptide (50 μM) surprisingly reduced the density of NeuN-expressing cells (i.e., those associated with mature neurons) in the midbrain of treated animals. Figure 2 and 3 As shown, administration of the T30 peptide induced a highly significant, dose-dependent increase in τ protein in all four brain regions studied. The highest dose tested (i.e., 100 μM) showed no difference in τ protein concentration compared to a control group injected with PBS. While not wishing to be bound to any hypothesis, the inventors believe this dose-dependent effect may be due to the closure of calcium channels upon excessive stimulation. However, at lower doses (i.e., less than 100 μM), where enhanced calcium inflow is active, the T30 peptide induces activation of glycogen synthase kinase 3 (GSK3), which leads to increased phosphorylation of τ protein, which in turn promotes the formation of τ protein tangles in the brain, a major marker of AD. In other words, the inventors have surprisingly shown that lower μM doses of T30 (i.e., less than 100 μM) are clearly receptor-mediated, while higher doses (i.e., above 100 μM) are not, which is entirely unexpected. Therefore, the inventors consider the dosage range of 0.1-99 μM T30 peptide or its fragments or variants to be optimal and therefore preferred, within which it is receptor-mediated.

[0050] The peptide, or its variants or fragments, can be introduced into the basal forebrain region of the brain. The peptide, or its variants or fragments, can be introduced into the septal / oblique band of the Broca (SID13) region of the brain. The peptide, or its variants or fragments, can be introduced into the cortical cholinergic system. Both the cortical and hippocampal septal cholinergic systems contribute to memory and are therefore preferred sites for peptide administration. However, preferably, the peptide, or its variants or fragments, can be introduced into the basal large cell nucleus (NBM).

[0051] Peptides can be administered to anesthetized animals via stereotactic injection, although administration to conscious animals via an implanted cannula may sometimes be preferred, for example, to examine acute effects (30-minute duration) without anesthesia. Alternatively, pressure microinjection or electrophoresis via (e.g., glass) micropipette may be preferred for ionophoretic recording.

[0052] Preferably, the non-human animal is a normal wild-type non-human animal. For example, the animal can be a mammal, which can be a primate, such as a monkey. The non-human animal can be male or female. However, preferably, the non-human animal is a rodent, which can be a mouse or a rat. Preferably, the rodent is a rat. The rat can be a Lister turban rat or a Long Evans turban rat. The rat can be male or female, but is preferably male. The rat can be an adult rat, i.e., at least 2 or 3 months old. Preferably, the non-human animal is a normal wild-type rodent.

[0053] Preferably, the peptide, or its variants or fragments, contribute to or induce neurodegeneration. The peptide, or its variants or fragments, administered to an animal model preferably induce cellular degeneration, thereby causing testable impairment of brain function, wherein impairment of the same brain function in humans represents neurological disorder.

[0054] For example, the model or method described herein can be used to study any neurodegenerative disease characterized by τ protein lesions. Neurodegenerative diseases can be selected from the group consisting of: Alzheimer's disease; Parkinson's disease; motor neuron disease; spinocerebellar types 1, 2, and 3; amyotrophic lateral sclerosis (ALS); Lewy body dementia; and frontotemporal dementia. Preferably, the model is used to study any neurological disorder related to the non-enzymatic function of acetylcholinesterase, particularly Alzheimer's disease, Parkinson's disease, and motor neuron disease.

[0055] However, models or methods are particularly preferred for studying Alzheimer's disease. Therefore, the testable brain function (whose impairment can be tested) can be cognitive function. Alternatively or additionally, this impairment can be attention deficit. Preferably, the method includes, for example, testing impairment of appropriate brain function in an animal model by providing the animal with an attentional task to test for attentional impairment.

[0056] Peptide treatment can be used to test for one or more impairments in memory, learning, attention, and / or problem-solving. A preferred method for testing cognitive function in animals is to perform spatial memory tests, such as the T-maze test (Rawlins et al., 1982, Beh, Brain Res, 5, 331-358). Other standard tests that can be used include the Morris water maze (Morris et al., 1982, Nature, 297, 681-683) and the radial arm maze (Olton et al., 1976, Animal Beh, Proc. 2, 97-116).

[0057] Preferably, the method involves combining the generation of peptide lesions in the brain (e.g., the basal forebrain) with the use of a device to test for attention deficits, which provides a series of selective response tasks. Rats can be trained to perform simple attentional tasks, such as pushing a panel with their noses to retrieve a food reward when a light flashes behind them. While sensitive to treatments affecting attention, failure to respond in such tests may also be due to the effect on performance. The treatment can, for example, induce sedation. The series of selective response tasks addresses this by providing more than one stimulus event; for example, a rat's lever press can result in one of three events: a flash of light from the left or right bin, or no light, in which case the correct choice is the central bin. Suitable devices for testing attention deficits in this manner have been described in Higgs et al., European J. Neuroscience (2000) 12, 1781-1788.

[0058] Other behavioral functions that can be monitored include, but are not limited to, social behavior, emotional responsiveness, situational regulation, pre-pulse inhibition of the startle reflex, bidirectional aversion regulation, and motivation measured by food and water intake or sucrose preference.

[0059] As noted above, it has been found that subtle lesions in the rat brains that induce attention deficits can be achieved using the peptide of SEQ ID NO: 3. However, it is envisioned that functionally equivalent lesions in the NBM can be achieved using other peptides as discussed above.

[0060] The animal models and methods described herein can be used to examine many molecular processes associated with τ protein lesions and related neurodegenerative disorders, test pharmacological compounds, and provide a reliable tool for drug screening of pharmacological compounds that modulate these processes.

[0061] Therefore, preferably, the method further includes administering the test reagent before, simultaneously with, or after the peptide or its variants or fragments, and determining whether the reagent can inhibit, prevent, or increase damage to the testable brain function and / or can inhibit, prevent, or increase cell damage in the brain. Preferably, the test reagent is selected as a compound capable of inhibiting or preventing damage to the testable brain function. Preferably, the method further includes synthesizing the test compound.

[0062] Therefore, in a third aspect of the invention, a non-human animal model prepared according to the second aspect or the method of the first aspect is provided for use in: (i) examining neurodegeneration or neuroregeneration processes; (ii) testing pharmacological compounds that can modulate neurodegeneration or neuroregeneration processes; or (iii) screening for neurodegeneration or neuroregeneration drugs.

[0063] Regulation of neurodegeneration can include inhibiting, preventing, or increasing neurodegeneration.

[0064] In a fourth aspect, a method for identifying candidate reagents for treating, preventing, or improving neurodegenerative disorders is provided, the method comprising:

[0065] - Administering the candidate reagent prepared according to the second aspect or the method according to the first aspect to an animal model; and

[0066] - To determine whether the candidate reagent inhibits, prevents, or increases impairment of testable brain function and / or causes improvement or worsening of cellular damage in the brain.

[0067] In this context, the ability to inhibit or prevent damage to testable brain function, or to improve cellular damage in the brain, indicates that the test reagent is a candidate for the treatment, prevention, or improvement of neurodegenerative disorders. Conversely, the ability to increase damage to testable brain function or to worsen cellular damage in the brain indicates that the reagent is not a candidate for the treatment, prevention, or improvement of neurodegenerative disorders.

[0068] Cellular damage can include neurodegeneration. This damage can be monitored or assessed by measuring one or more of the following:

[0069] (i) Inhibition of activity in a population of neurons (i.e., a set);

[0070] (ii) Calcium levels;

[0071] (iii) The activity level of acetylcholinesterase;

[0072] (iv) Expression of α-7 nicotinic receptors in the cell membrane; and

[0073] (v) The cell density and / or loss or reduction of NeuN-expressing cells in specific regions (associated with neuronal death).

[0074] Preferably, the testable brain function can be cognitive function or attention deficit. Preferably, the method includes damage to an experimental animal model or cognitive function or attention deficit.

[0075] In the fifth aspect, a method is provided for testing the bioactivity of an experimental reagent in neurodegenerative diseases, wherein the method comprises administering the experimental reagent to an animal model prepared according to the second aspect or by the method of the first aspect, and assessing any changes, improvements or deteriorations in the animal with brain injury related to the brain injury.

[0076] This assessment will include determining whether the reagent will inhibit, prevent, or increase impairment of appropriate testable brain functions, such as cognitive functions like attention or memory, and / or determining whether there is any improvement or deterioration in cell damage at relevant sites in the brain. The test reagent is preferably a pharmaceutical compound.

[0077] The following is a list of some other behavioral tests suitable for use according to the present invention. Most, but not all, of these are tests of cognitive function. Tests involving behavior but not cognitive ability are also included, and these tests may be used in place of tests of cognitive function (such as memory), or in addition to tests of cognitive function (such as memory).

[0078] attention

[0079] Carli, M., Robbins, TW, Evenden, JL, and Everitt, BJ (1983) Effects of lesions toascending noradrenergic neurons on performance of a 5-choice serial reactiontime task in rats—implications for theories of dorsal noradrenergic bundle function based on selective attention and arousal (Behavioural Brain Research 9, 361-380).

[0080] social behavior

[0081] Gardner, CR and Guy, AP (1984) on acutely administered benzodiazepines A social interaction model of anxiety sensitive to acutely administered benzodiazepines (Drug Dev.Res.4, 207216).

[0082] Emotional reactivity

[0083] Gray, JA (1982) The neuropsychology of anxiety. Dawson, GR and Tricklebank MD (1995) Use of the elevated plus maze in the search for novel anxiolytic agents. TIPS 16, 33-36.

[0084] Morris Water Maze

[0085] Morris, RGM, Garrud, P., Rawlins, JNP, and O”Keefe, J. (1982) Impaired place navigation in rats with hippocampallesions, Nature 297, 681-683.

[0086] Radial arm maze

[0087] Olton, DS and Samuelson, RJ (1976), Remembrance of places past: 20 spatial memories in rats, Journal of Experimental Psychology: Animal Behaviour Processes 2, 97-116.

[0088] T-shaped maze

[0089] Rawlins, JNP and Oiton, DS (1982) The septo-hippocampal system and cognitive mapping (Behavioural Brain Research 5, 331-358).

[0090] It will be appreciated that the invention extends to any nucleic acid or peptide, or its variants, derivatives, or like thereof, comprising a substantially amino acid or nucleic acid sequence of any sequence mentioned herein, including its functional variants or functional fragments. The terms "substantially amino acid / nucleotide / peptide sequence," "functional variant," and "functional fragment" can mean an amino acid / nucleotide / peptide sequence having at least 40% sequence identity with any of the sequences mentioned herein, for example, a sequence having 40% identity with the sequence identified herein.

[0091] Amino acid / polynucleotide / peptide sequences having sequence identity greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% with any of the mentioned sequences are also envisioned. Preferably, the amino acid / polynucleotide / peptide sequence has at least 85% identity with any of the mentioned sequences, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99%, i.e., SEQ ID NO: 1-5.

[0092] Technicians will understand how to calculate the percentage identity between two amino acid / polynucleotide / peptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / peptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The percentage identity of the two sequences can take different values ​​depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used in the alignment method, such as local-to-global alignment, the matrix of pair scores used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, such as functional forms and constants.

[0093] After alignment, there are many different ways to calculate the percentage identity between two sequences. For example, one can divide the amount of identity by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-empty positions; or (v) the number of equivalent positions excluding protruding ends. Furthermore, it will be recognized that percentage identity is also strongly dependent on length. Therefore, the shorter a pair of sequences, the higher one can expect to be of coincident sequence identity.

[0094] Therefore, it will be recognized that accurate alignment of protein or DNA sequences is a complex process. The popular multiplex alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiplex alignments of proteins or DNA according to the present invention. Suitable parameters for ClustalW can be as follows: For DNA alignment: vacancy opening penalty = 15.0, vacancy extension penalty = 6.66, and matrix = identity. For protein alignment: vacancy opening penalty = 10.0, vacancy extension penalty = 0.2, and matrix = Gonnet. For both DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will recognize that these and other parameters may need to be modified for optimal sequence alignment.

[0095] Preferably, the percentage identity between two amino acid / polynucleotide / peptide sequences can then be calculated from an alignment such as (N / T)*100, where N is the number of positions where the sequences share the same residues, and T is the total number of positions compared, including vacancies and including or excluding overhangs. Preferably, overhangs are included in the calculation. Therefore, the most preferred method for calculating the percentage identity between two sequences includes: (i) preparing the sequence alignment using the ClustalW program with a suitable set of parameters, for example, as described above; and (ii) inserting the values ​​of N and T into the following equation: - Sequence Identity = (N / T)*100.

[0096] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, substantially similar nucleotide sequences will be encoded by sequences that hybridize with DNA sequences or their complementary sequences under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filtered-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at least once in 0.2x SSC / 0.1% SDS at about 20-65°C. Alternatively, substantially similar polypeptides may differ from the sequences shown in SEQ ID Nos: 1-5 by at least 1, but less than 5, 10, 20, 50, or 100 amino acids.

[0097] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or altered without substantially affecting the protein sequence it encodes, thus providing functional variants. Suitable nucleotide variants are those altered by different codon substitutions that encode the same amino acid within the sequence, thus producing a silent alteration. Other suitable variants are those with homologous nucleotide sequences, but containing all or part of the sequence, altered by different codon substitutions that encode amino acids with side chains having similar biophysical properties to the amino acid it substituted, thus producing conserved alterations. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it will become apparent which amino acids can be replaced by amino acids with similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0098] All features described herein (including any appended claims, abstracts and drawings), and / or all steps of any disclosed methods and processes may be combined with any of the foregoing aspects in any combination, except that at least some of such combinations of features and / or steps are mutually exclusive.

[0099] To better understand the present invention, and to illustrate how embodiments of the present invention can be implemented and effective, reference will now be made to the accompanying drawings by way of example, wherein:

[0100] Figure 1 The levels of β-amyloid protein (42) in the dissected rat brain (cortex, subcortex, hippocampus and cerebellum) were shown after PBS (control) or 1 μM, 50 μM or 100 μM T30 (treatment) was injected into the basal forebrain.

[0101] Figure 2 The levels of total τ protein in the dissected rat brain (cortex, subcortex, hippocampus, and cerebellum) are shown after injection of PBS (control) or 1 μM, 50 μM, or 100 μM T30 (treatment) into the basal forebrain.

[0102] Figure 3 The percentage of τ protein in dissected rat brain regions (cortex, subcortex, hippocampus, and cerebellum) is shown after PBS (control) or 1 μM, 50 μM, or 100 μM T30 (treatment) is injected into the basal forebrain.

[0103] Figure 4 The levels of T14 in the dissected rat brain (cortex, subcortex, and cerebellum) were shown after PBS (control) or 1 μM, 50 μM, or 100 μM T30 (treatment) was injected into the basal forebrain.

[0104] Figure 5 Data taken from a paper (Garcia Rates et al., 2016, “Pharmacological profiling of a novel modulator of the α7nicotinicreceptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains”, Neuropharmacology, 2016, Jun, 105:487-499) shows that lower doses of T30 lead to calcium influx into PC12 cells, which in turn causes an increase in glycogen synthase kinase 3 (GSK3) levels.

[0105] Figure 6 This illustrates the cascade of events resulting from the effects of T30 in the cell;

[0106] Figure 7 Immunohistochemical staining of sections from WT SD rats after acute treatment with T30 peptide or saline. Intracellular pτ (yellow) was not detected in the hippocampus, cortex, midbrain, basal forebrain, or pons / medulla. NeuN (green) was used to detect neurons, and the nucleus was detected with DAPI (blue).

[0107] Figure 8 This study presents a quantitative analysis of the effects of AChE-derived peptide (T30) on Alzheimer's disease-related parameters (pτ, NeuN) in SD rats. The density of NeuN-positive cells in the cortex, hippocampus, midbrain, pons / medulla, and basal forebrain of WT SD rats was quantified after treatment with T30 peptide or saline. Statistical analysis was performed using an unpaired t-test. *p<0.05; **p<0.01 Saline control; n=8; T30 peptide, n=8; 6 slices per animal;

[0108] Figure 9The IHC effect of AChE-derived peptide (T30) on Alzheimer's disease-related parameters (6E10, Iba1) in SD rats is shown. Immunohistochemical staining of sections from WT SD rats after treatment with T30 peptide or saline is shown. No intracellular Aβ deposits (yellow) were detected in the hippocampus, cortex, midbrain, basal forebrain, and pons / medulla. Microglia were detected with Iba1 (green), and cell nuclei were detected with DAPI (blue).

[0109] Figure 10 This study presents a quantitative analysis of the effects of AChE-derived peptide (T30) on Alzheimer's disease-related parameters (pτ, NeuN) in SD rats. The density of Iba1-positive cells in the cortex, hippocampus, midbrain, pons / medulla, and basal forebrain of WT SD rats was quantified after treatment with T30 peptide or saline. Statistical analysis was performed using an unpaired t-test. *p<0.05; **p<0.01 Saline control; n=8; T30 peptide, n=8; 6 slices per animal;

[0110] Figure 11 The results of the Morris Water Maze (MWM) experiment at quadrant and plateau time point 1 are shown;

[0111] Figure 12 The results of the Morris Water Maze (MWM) quadrant and plateau time point 2 experiment are shown;

[0112] Figure 13 The results of the Morris Water Maze (MWM) experiments at quadrants and plateau time points 1, 2, and 3 are shown.

[0113] Figure 14 This demonstrates chronic impairment of memory in the water maze induced by T30 over time; and

[0114] Figure 15 The effects of a single intracerebral injection of T30 on normal rats were shown at 3, 16, and 24 weeks. Specific Implementation

[0115] Despite studies on the increased number of major events in neurodegeneration, no animal model closely replicates the full pathological profile (e.g., the pathological profile of Alzheimer's disease) because the fundamental mechanisms of neurodegeneration remain poorly understood. Therefore, the inventors have developed a novel in vivo animal model to elucidate the fundamental mechanisms inducing neurodegeneration, and importantly, in which new experimental reagents can be tested to determine the activity of neuroprotection (or neurotoxicity).

[0116] This invention relates to the use of peptide T30 (SEQ ID N: 3), a peptide cleaved from the C-terminus of acetylcholinesterase (AChE), which consists of a biologically active 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 demonstrated the application of AChE-derived peptides in cell lines to promote an AD-like phenotype. These effects were blocked by NBP-14, a novel candidate regulator of α7-nAChR, which is a cyclic form of T14 and therefore has the cyclic sequence SEQ ID No: 4. As described below, the inventors have applied T30 or NBP14 to brain slices from in vivo and investigated their activity in regulating endogenous T14 expression and whether they contribute to or prevent neurodegeneration patterns.

[0117] The inventors demonstrate that the device and model can be used to study neurodegeneration under more physiological conditions, namely, the expression of p-τ and Aβ on α7-nAChR from brain slices in vivo, although it will be recognized that there are any other proteins that can be measured to monitor the degree and progression of neurodegenerative disorders. The model utilizes a novel hypothesis that the inventors believe explains the aberrant processes characterizing AD, based on the interaction between the α7 nicotinic acetylcholine receptor (α7-nAChR) and a toxic peptide (i.e., T30) cleaved from the C-terminus of acetylcholinesterase (AChE). The device and model can be used to examine numerous molecular processes, test pharmacological compounds, and provide a reliable tool for drug screening, thereby reducing the need for animal experiments where the pharmacological compound can modulate these processes.

[0118] Materials and methods

[0119] Brain extraction and dissection

[0120] Following a lethal injection of anesthesia (pentobarbital), fresh brains were extracted, and the cortex, hippocampus, cerebellum, and inferior cortex were dissected and immediately rapidly frozen in liquid nitrogen. The brains were stored at -80°C to preserve proteins. Due to the death of one rat prior to the start of the experiment, the groups were as follows:

[0121] PBS (control): n=6

[0122] 1μM T30: n=5

[0123] 50μM T30: n=6

[0124] 100μM T30: n=6

[0125] Brain homogenization

[0126] Thaw brain slices on ice and add ice-cold lysis buffer (PBS + protease and phosphatase inhibitors, 1:100 each) to each slice. Using a mortar and pestle, homogenize the tissue as much as possible before using the sonicator probe at low speed, for 5 seconds at a time, while keeping on ice, until the tissue is completely homogenized. Incubate the samples on ice for 20 minutes, then centrifuge at 4°C at maximum speed (13,000 rpm) for 30 minutes. Remove the supernatant and use it for analysis.

[0127] β-Amyloid ELISA

[0128] Purchase a commercial ELISA kit for β-amyloid protein 42 (Invitrogen, KMB3441) and β-amyloid peptide (1-42) (Abcam, ab120959) together. Plate all samples with 6000 μg / mL total protein (determined by Pierce protein assay) plus a positive control synthesized from wild-type whole rat brain, plus 275 ng (the publicly known concentration found in transgenic animal models of AD) of β-amyloid peptide (1-42). A second control (without the primary antibody) and a chromogenic blank were also used on each plate. A standard curve of β-amyloid peptide (1-42) ranging from 0–200 pg / mL was prepared on each plate according to the protocol described in the kit (except for the peptide supplied with the kit, which was replaced by the alternatives listed above). Generally, standards (in duplicate) and samples (in triplicate) were plated and incubated at room temperature on a plate shaker for 2 hours. Aspirate all standards and samples, and wash the plate before adding the "detection" antibody provided with the kit to all wells (except the second control and chromogenic blank). Incubate on a plate shaker at room temperature for another 1 hour cycle, then aspirate the antibody and wash the plate again. Add IgG HRP to each well (except the chromogenic blank) and incubate on a plate shaker at room temperature for another 30 minutes. Aspirate all solutions and wash the plate, then add the stable chromogenic antibody to each well and incubate on a plate shaker at room temperature in the dark for 30 minutes. Finally, add the stop solution to each well and read the absorbance at 450 nm.

[0129] Total t-protein ELISA

[0130] A commercial ELISA kit (Abcam, ab210972) for total τ protein detection was purchased. All samples were plated with 0.5 μg / mL total protein (determined by the Pierce protein assay) and τ protein from the full standard curve ranging from 0-2000 pg / mL, along with a second control (without the first antibody). Following the protocol described in the kit, approximately, standards (two copies) and samples (three copies) were plated, followed immediately by the addition of the antibody mixture (minus the capture antibody of the second control), and incubated on a plate shaker at room temperature for 1 hour. All solutions were aspirated and the plate was washed, then TMB substrate was added to all wells and incubated on a plate shaker in the dark at room temperature for 10 minutes. Finally, stop solution was added to all wells, and the plate was incubated on a plate shaker at room temperature for 1 minute, at which point the absorbance was read at 450 nm.

[0131] T14 ELISA

[0132] The inventors have developed an internal ELISA for detecting T14. All remaining samples (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 with a full T14 standard curve (duplicates) ranging from 0-40 nM and a second control plate (triples). The plates were incubated overnight at 4°C on a shaker, and then completely aspirated before adding the BSA blocking solution and further incubated at room temperature on a plate shaker for 6 hours. Aspirate the blocking solution and add the first antibody (T14 polyclonal, Genosphere) to all wells (except the second control), and incubate overnight at 4°C on a plate shaker. Aspirate the antibody solution and wash the plate, then add the second antibody and incubate at room temperature on a plate shaker for 2 hours. Aspirate all the solution and wash the plate, then add the TMB substrate and incubate the plate at room temperature on a plate shaker for 15 minutes. Add the stop solution and read the absorbance at 450 nm.

[0133] Tissue preparation and immunohistochemistry

[0134] Mouse brain samples were removed from PBS and cryoprotected by incubation in 30% sucrose solution for 72 hours or until saturation. The whole brain was cut in half along the midline, and each half was embedded in TissueTek and stored at -80°C until the time for frozen sectioning.

[0135] Starting at the midline, cut 25 μm sagittal sections using a cryostat. Collect the sections in 24-well plates and use them directly for staining or store them in a -20°C cryoprotectant solution (25 mM sodium phosphate buffer, pH 7.4, 30% ethylene glycol, 20% glycerol) until use. Stain all sections mounted on ultra-rough slides.

[0136] Immunostaining for the detection of β-amyloid protein (Aβ), phosphorylated τ protein (pτ), neurons (NeuN), and microglia (Iba1) was performed as follows. Section pretreatment was performed for antigen retrieval: either for pτ, in citrate buffer (pH 6.0) at 90°C for 30 minutes, or for Aβ, in 70% formic acid for 10 minutes. After infiltration into 0.3% Triton X-100 / PBS, the antigen-retrieval sections were blocked in 10% normal goat serum / PBS and incubated overnight at 4°C with a primary antibody diluted in 0.1% Triton X-100 in 1% normal goat serum and PBS.

[0137] The following primary antibodies were used for immunostaining: anti-β-amyloid (Aβ) monoclonal mouse, 6E10, (1:1000; Covance, cat#39320), anti-phosphorylated τ protein monoclonal mouse, AT180, (1:500; Thermo, cat#MN1040), anti-Iba1 monoclonal rabbit (1:500; Synaptic System, cat#234004), and anti-NeuN polyclonal rabbit (1:500; Millipore, cat#ABN78).

[0138] Co-staining was performed using 6E10 in combination with Iba1 and AT180 in combination with NeuN. Sections were washed three times in PBS for 15 minutes each time and incubated at room temperature in a suitable secondary antibody (Sigma) for 2 hours. Sections were washed three more times in PBS for 15 minutes each time, then stained with DAPI and incubated to detect cell nuclei. Finally, mounting medium was applied to the stained sections, and slides were placed on top of coverslips for imaging using the Zeiss AxioScan.Z1 system (Carl Zeiss Microscopy).

[0139] Image acquisition and quantitative analysis

[0140] Automated image acquisition was performed using a Zeiss AxioScan.Z1 slide scanning system (Leica Biosystems) equipped with an LED-Colibri7 light source and an Axiocam 506 single-camera system. Images were captured non-confocal at 20x magnification (pixel size: 0.22μm) and visualized using Zen software. The image data was then input into... In the image analysis software (Visiophoarm A / S), region selection is performed.

[0141] Manual segmentation of images of sagittal brain slices was performed using coordinates published by Allen Developing Mouse Brain Atlas (Allen Institute) as a guide, to subdivide the cortex, hippocampus, midbrain, basal forebrain, and pons / medulla regions.

[0142] use Studio 5.1 (PerkinElmer Inc.) and its integration Batch analysis as As part of the system, image analysis scripts were developed for the characterization and quantification of intracellular and extracellular Aβ, pτ, NeuN, and Iba1. For all analyses, DAPI signals and based on… The "Nuclear Detection B" algorithm's customized nuclear detection workflow identifies individual cells within tissue sections. Several quality control parameters are implemented to discard out-of-focus nuclei and non-nuclear structures. These include, for example, applying thresholds for minimum signal contrast, nuclear area, and nuclear roundness. The cytoplasm of the detected cells is defined as a 4-pixel-wide concentric ring surrounding the previously segmented nucleus (perinuclear region). Outside this perinuclear ring, a 3-pixel-wide background region is generated as the individual cell, and after median aggregation, a whole-brain region reference area is used to determine the NeuN- and Iba1-positive cell populations.

[0143] The signal intensity of Aβ, pτ, NeuN, and Iba1 staining was evaluated in all cellular subregions. Cells were identified as NeuN or Iba1 positive when the mean signal intensity in the nuclear region was at least 1.5 or 2 times higher than the intermediate background of the brain region, respectively.

[0144] Extracellular plaques were segmented by applying intensity thresholds to the images: signals with an intensity at least twice that of the amyloid protein background in intermediate cells were considered potentially belonging to plaques. To exclude false-positive plaques from the analysis, these initial plaque-like targets were further filtered by applying thresholds for minimum plaque size (i.e., >200 px²) and axial ratio (minor axis length / major axis length >0.4). All readings were calculated as averages for each brain region and histological section. These values ​​were then used to calculate individual averages for each animal.

[0145] Data processing and analysis

[0146] A total of 16 animals were used in the study, with N = 8 animals in each treatment group. Quantitative results were obtained from an average of six slides per animal to produce one data point per animal. Statistical analysis was performed using an unpaired t-test. *p<0.05; **p<0.01 T30 peptide versus saline.

[0147] Antibodies for immunohistochemical analysis of brain samples from SD rats

[0148] AD-related pathology Detected phenotype First Antibody Aβ plaques β-amyloid protein 6E10 τ protein Phosphorylated τ protein AT8 Glial hyperplasia Active microglia Iba1 Cell loss Neuron cell count NeuN

[0149] analyze

[0150] First, from the absorbance value (A) 450 Calculate the standard deviation of the blank, the limit of detection (LOD) (standard deviation of blank x 3.3), and the limit of quantitation (LOQ) (standard deviation of blank x 10). If applicable, subtract the mean of the chromogenic blank from all standard curves, sample, and control values, then subtract the mean of the blank, and then subtract the mean of the second control. Using GraphPad Prism software, plot all values ​​above the LOQ, and interpolate values ​​(if applicable) to pg / mL. Perform all statistical analyses using GraphPad Prism software.

[0151] Simple-Step ELISA Kit for Human τ Protein – Abcam ab210972:

[0152] The plan is as follows:

[0153] - Prepare all reagents, working standards and samples.

[0154] Remove excess micro-strips from the plate frame, return them to the foil pouch containing the desiccant, reseal, and return to 4°C for storage.

[0155] Add 50 μl of all samples or standards to the appropriate wells.

[0156] Add 50 μl of the antibody mixture to each well.

[0157] - Seal the plate and incubate it at room temperature for 1 hour on a plate shaker set to 400 rpm.

[0158] - Wash each well with 3 x 350 μl 1X Wash Buffer PT. Wash by aspiration or decanting from the wells, then dispense 350 μl 1X Wash Buffer PT into each well. Complete removal of liquid is important for good performance at each step. After the final wash, invert the plate and blot it dry on a clean paper towel to remove excess liquid.

[0159] Add 100 μl of TMB substrate to each well and incubate in the dark for 10 minutes on a plate oscillator set to 400 rpm.

[0160] Add 100 μl of the stop solution to each well. Shake the plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm, and this is...

[0161] In addition, a second control was added to all plates, and during normalization, all A... 450 Subtract these second controls from the values.

[0162] - For peptides in the standard curve, dilute the standard and all samples in 1X cell extraction buffer (5X cell extraction buffer PTR provided in the kit) plus 1X cell extraction enhancer solution (50X cell extraction enhancer solution provided in the kit) in dH2O.

[0163] - Prepare 1x wash buffer by diluting 10x wash buffer PT (provided in the kit) with dH2O.

[0164] -Antibody mixture:

[0165] ο1x human τ protein capture antibody + 1x human τ protein detection antibody (both in 10x form as provided in the kit) are being diluted.

[0166] - Antibody used as a second control:

[0167] ο Dilute 1x human τ protein detection antibody (10x form provided by the kit) with antibody diluent CPI (provided in the kit).

[0168] Statistical analysis:

[0169] -From all standards and samples A 450 The average A of the values ​​minus the blanks 450 The average value;

[0170] -From all standards and samples A 450Subtract the second control's A from the value 450 The average value;

[0171] -Use Dunnett's multiple post-hoc comparison test to perform a standard one-way ANOVA on each brain region compared to a control for that region.

[0172] Morris's Water Maze Method

[0173] A 2.1m diameter black water maze pool was filled to a depth of 40cm with 22°C water. This placed a 15cm diameter submersible platform 1cm below the water level. Rats were then placed in the water at a datum point (N, E, S, W) quadrant and allowed 2 minutes to find the platform. If the rat found the platform within that time, it was allowed to remain on it for 15 seconds, gently wrapped in a towel, and placed under a heat lamp. It was then removed. If the rat did not find the platform within 2 minutes, it was guided to the platform by dragging its hand in the water in front of it. It was then allowed to remain on the platform for 15 seconds, wrapped in a towel, and placed under a heat lamp before being removed. This routine was repeated 4 times daily (up to 10 days, although the current citation allows for 6 days of trials, including 4 days of reverse learning), until the rat had clearly learned the maze, as indicated by no significant improvement after 3 consecutive days. The interval between trials was 10 minutes, and a probe test was performed at the end of both the reference memory test and the reverse learning test to probe working memory.

[0174] Example 1

[0175] The primary objective was to determine whether a single dose of T30 injected into the basal forebrain of WT rats could neurochemically induce an "Alzheimer's-like" profile, defined as a statistically significant increase in AD-related proteins in the treatment group compared to the control. Secondly, this work aimed to determine at what concentration of T30 these changes were elicited.

[0176] At the University of Nottingham, stereotactic injection of either PBS (control) or one of three doses of T30 (1 μM, 50 μM, and 100 μM) into the MS / VDB (septum / vertical branch of the oblique band) of adult male Listerian hooded rats was performed. Rats were selected 2–3 weeks post-injection, and the brain was extracted and dissected to separate the cortex, hippocampus, cerebellum, and inferior cortex regions for neurochemical analysis in neurobiology. The levels of total τ protein, β-amyloid protein 42, and T14 were analyzed in each brain region.

[0177] result

[0178] Example 1: β-Amyloid protein (42)

[0179] refer to Figure 1Due to the difficulty in detecting β-amyloid protein 42 in samples, the number of values ​​above the limit of quantitation (LOQ) was small, and subsequently, not all brain regions and doses could be statistically analyzed. From those values ​​above the LOQ, compared with the PBS control, there was no statistically significant effect on T30 in any brain region at any dose (1 μM: cortex p = 0.8843, subcortex p = 0.8138, hippocampus p = 0.8494, cerebellum p = insufficient data point; 50 μM: cortex p = 0.7794, subcortex p = 2086, hippocampus p = 0.2253, cerebellum p = insufficient data point; 100 μM: cortex p > 0.9999, subcortex p = 0.7484, hippocampus p = 0.9975, cerebellum p = 0.8069) (see [link to relevant documentation]). Figure 1 ).

[0180] Note that all data showing β-amyloid protein 42 are normalized to the positive control, not in pg / mL. Determining pg / mL would provide inaccurate quantification due to the difficulty of the assay, and therefore represent unreliable data.

[0181] Example 2: Total τ protein

[0182] refer to Figure 2 Compared with the PBS control, T30 at concentrations of 1 μM and 50 μM significantly increased total tau protein levels in all brain regions (1 μM: cortex p = 0.0186, subcortex p = 0.0003, hippocampus p = 0.0015, cerebellum p = 0.0052; 50 μM: cortex p = 0.0339, subcortex p = 0.0042, hippocampus p = 0.0409, cerebellum p = 0.0104). Compared with the PBS control, at the highest dose of T30 (100 μM), there was no significant difference in total tau protein levels in any brain region (cortex p = 0.8976, subcortex p = 0.9824, hippocampus p = 0.6805, cerebellum p = 0.5228). Figure 2 ).

[0183] refer to Figure 3 This allows us to see the percentage of τ protein in the dissected rat brain regions.

[0184] (i) 1 μM T30: led to a 50% increase in tau protein in the cortex, a 90% increase in tau protein in the subcortex, a 60% increase in tau protein in the hippocampus, and an 80% increase in tau protein in the cerebellum.

[0185] (ii) 50 μM T30: led to a 45% increase in tau protein in the cortex, a 70% increase in tau protein in the subcortex, a 40% increase in tau protein in the hippocampus, and a 70% increase in tau protein in the cerebellum.

[0186] Example 3-T14

[0187] Reference Figure 4 Compared with the control (PBS), there were no significant differences in T14 levels at any concentration of T30 (1 μM, 50 μM, or 100 μM) in any brain region analyzed (cortex, subcortex, cerebellum) (1 μM: cortex p = 0.3670, subcortex p = 0.7354, cerebellum p = 0.1273; 50 μM: cortex p = 0.9917, subcortex p = 0.9996, cerebellum p = 0.9952; 100 μM: cortex p = 0.8740, subcortex p > 0.9999, cerebellum p = 0.6297). Figure 3 It is worth noting that a limited number of samples were reserved for T14 analysis. No hippocampal samples were reserved for further testing.

[0188] Example 4 - Effects of AChE-derived peptide (T30) on Alzheimer's disease-related parameters (pτ, NeuN) in SD rats ring

[0189] As described in these methods, sagittal brain sections from SD rats that received acute administration of T30 peptide or saline were prepared using a cryostat. Every sixth section was collected starting from the midline, and six sections from each animal were immunostained for the detection of Aβ (6E10), pτ (pS202 / pT205), microglia (Iba1), and neurons (NeuN). For all animal samples, the primary antibody was combined in two co-stained groups. Quantitative analysis of different markers was performed in five distinct target regions (ROIs), including the cortex, hippocampus, basal forebrain, midbrain, and pons / medulla.

[0190] refer to Figure 7 Immunohistochemical staining of sections from WTSD rats following acute treatment with T30 peptide or saline. Intracellular pτ (yellow) was not detected in the hippocampus, cortex, midbrain, basal forebrain, or pons / medulla. NeuN (green) was used to detect neurons, and DAPI (blue) was used to detect cell nuclei. Therefore, immunohistochemical results show that intracellular pτ (pS202 / pT205) protein was not detected in any stained brain sections from SD rats treated with T30 peptide or saline in the cortex, hippocampus, midbrain, basal forebrain, or pons / medulla (see [link to immunohistochemical staining]). Figure 7 Interestingly, compared with saline-treated animals, a significant reduction in the density of NeuN-positive cells was observed in the midbrain of SD rats after administration of T30 peptide (see [link to article]). Figure 7 ).

[0191] refer to Figure 8This paper presents the quantification of NeuN-positive cell density in the cortex, hippocampus, midbrain, pons / medulla, and basal forebrain of WT Sprague Dawley rats after treatment with T30 peptide or saline. As can be seen, no difference in NeuN-positive cell density was observed in other brain regions, including the cortex, hippocampus, and basal forebrain, although a decreasing trend was observed in the pons / medulla region (see [link to original text]). Figure 8 ).

[0192] Example 5 - Effects of AChE-derived peptide (T30) on Alzheimer's disease-related parameters (6E10, Iba1) in SD rats Influence

[0193] Sagittal brain sections from SD rats were prepared, and IHC was performed in a second co-staining group to detect 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. Figure 8 ).

[0194] Furthermore, no difference in the total number or density of Iba1-positive cells was observed in the cortex, hippocampus, cortex, midbrain, basal forebrain, or pons / medulla (see [link to relevant documentation]). Figure 8 ).

[0195] Example 6 - Animal Model Behavioral Research

[0196] 1) Morris Water Maze Time Point 1

[0197] Both the 6-day MWM learning curve and the further 4-day reverse learning curve revealed no significant differences between the treatment groups on any day. Two-way ANOVA (genotype x day) with repeated measures was used. There were no significant differences between the treatment groups in the time spent in or accessing the target quadrant for both probe tests (PT) and reverse probe tests (RPT). Two-way ANOVA (genotype x quadrant).

[0198] However, reference Figure 11 Although there was no significant difference in the time spent at the target platform site or the time spent accessing the target platform site during PT, for the peptidomimetry, a significant reduction in the time spent at the target platform site was found during RPT (p = 0.011). Two-way ANOVA (genotype x platform) was performed. Furthermore, an interaction was found between genotype and platform (p = 0.014).

[0199] Although probe assays revealed good differentiation of the target quadrants in both treatment groups, this was less evident in the peptide group during reverse probe assays, which were used to access the target quadrants and target plateau regions. This was indicated by the lack of significant differences in access to the target plateau and quadrant regions compared to the regions previously targeted in the probe assays.

[0200] 2) Morris Water Maze Time Point 2

[0201] Both the MWM 4-day learning curve and the further 4-day reverse learning curve indicated no significant differences between the treatment groups on any given day. Two-way ANOVA (genotype x day) with repeated measures was performed. In both the probe test (PT) and reverse probe test (RPT), there were no significant differences between the treatment groups in the time spent in or accessing the target quadrant. Two-way ANOVA (genotype x quadrant).

[0202] refer to Figure 12 A significant difference was found in the time spent in the target plateau region during PT, but not during PT. Compared with the saline control, the peptide group revealed a reduction in time spent in the plateau region (p = 0.01). Furthermore, an interaction between genotype and plateau was found (p < 0.001). Interestingly, although a similar pattern of time spent in the plateau region was observed in RPT, this was not statistically significant. Upon closer examination, this appears to be due to one rat from the peptide group spending twice as much time in the plateau region during RPT. Rats in both PT and RPT revealed good differentiation of the target quadrant and plateau region in both treatment groups.

[0203] 3) Morris Water Maze Time Point 3

[0204] refer to Figure 13 No individual results from time point 3 were significant. However, when placed in the context of time points 1 and 2, an increasing trend in target plateau time was observed in the saline group, while the target plateau time in the T30 group tended to remain the same, indicating memory impairment in the T30 group (see [link to relevant documentation]). Figure 14 ).

[0205] Application of Example 6-T30

[0206] refer to Figure 15 The figure shows the effects of a single intracerebral injection of T30 on normal rats at 3, 16, and 24 weeks post-administration. As can be seen, the figure includes histology at intermediate time points and shows significant and marked cell loss in key brain regions (i.e., the brain regions primarily vulnerable in Alzheimer's disease), as well as significant decreases in adjacent regions from the same group of vulnerable cells.

[0207] in conclusion

[0208] Total τ protein

[0209] Surprisingly, intermediate doses (1 μM and 50 μM) of T30 peptide significantly increased total τ protein levels in all brain regions (cortex, subcortex, hippocampus, and cerebellum), with levels returning to control levels at the highest dose (100 μM). The 1 μM T30 dose showed the greatest increase in total τ protein levels across all regions.

[0210] β-Amyloid protein 42

[0211] Two to three weeks prior to rat sacrifice, no significant differences in β-amyloid protein levels were found in any region of the dissected brain (cortex, subcortex, hippocampus, or cerebellum) following a single injection of T30 peptide into the basal forebrain. Previous studies (Lin et al., 2009, J. Alzheimer's Dis, 18(4):907-18) have clearly established that increased total τ protein, rather than β-amyloid protein, in the CSF is associated with short-term memory impairment in Alzheimer's disease. The results described here are inconsistent with these earlier findings of unchanged β-amyloid protein levels, despite significantly elevated τ protein levels.

[0212] T14

[0213] Compared with controls, T14 levels did not differ significantly across any of the samples analyzed (cortex, subcortex, and cerebellum) at any concentration of T30. No hippocampal samples remained for T14 level analysis, and a limited number of other regions were also included.

[0214] NeuN positive cells

[0215] The density of NeuN-positive or expressing cells was significantly reduced in the midbrain, while no difference was observed in other brain regions (cortex, hippocampus, basal forebrain, or pons / medulla). NeuN levels indicate the number of mature neurons present.

[0216] Overview

[0217] As shown in the figure, T30 peptide treatment induced a highly significant, dose-dependent increase in τ protein in all four brain regions studied. In all cases, the highest dose (i.e., 100 μM) showed no difference from the control group injected with PBS. The inventors hypothesized that this highest dose was most likely due to the closure of calcium channels during overstimulation (Standen, 1981, "Ca inactivation by intracellular Ca injection into Helix neurons", Nature, 293, 158-159), as previously used in breast cancer cells (Onganer et al., 2006, "Anacetylacholithica-derived peptide inhibits endocytic membrane activity in a human transferative breast cancer cell line", Biochimica et Biophysica Acta, 1760(3): 415-420) and α7-transfected oocytes (Greenfield et al., 2004, "A novel peptide modulates α7 nicotinic receptor response: revealing possible mechanisms of nutritional toxicity in the brain"). alpha7nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain. (J Neurochem 90, 325-331) and in brain slices (Bon et al.).[References: 2003, Bioactivity of acetylcholinesterase-derived peptides: electrophysiological characterization in guanine-pig hemapus (Eur J Neurosci 17, 1991-1995); and high-dose peptides observed in organoid hippocampal neurons (Day and Greenfield 2004, A non-cholinergic, trophic action of acetylcholinesterase onhamamal neurons in vitro: Molecular mechanisms (Neuroscience 111, 649-656)).

[0218] However, at lower doses (less than 100 μM), where the enhanced calcium influx is active, the T30 peptide induces GSK activation (Garcia Rates et al., 2016, “Pharmacological profiling of a novel modulator of the α7nicotinic receptor: Blockade of a toxic acetylcholine-derived peptide increased in Alzheimer’s brain”, Neuroopharmacology, Vol. 105, pp. 487-499), which in turn leads to increased phosphorylation of the tau protein (Rankin et al., 2007, “Tau phosphorylation by GSK-3β promotes tangle-like filament morphology”, Mol Neurodegener). 2:12), which in turn promotes the formation of tangles, a major marker of AD (Braak and Braak 2011, "Stage of the pathological process in Alzheimer's disease: age category from 1 to 100 years", J. Neuropathol ExpNeurol. 70(11): 960-9). In other words, the inventors have surprisingly shown that low doses of T30 are receptor-mediated, while high doses are not, and this is completely unexpected. Therefore, the inventors believe that the 1-99 μM T30 dose range, in which receptors are mediated, is optimal and preferred.

[0219] Figure 6 This illustrates the cascade of events resulting from the effects of T30 in the cell;

[0220] (1) T30 binds to the allosteric site of the receptor to enhance Ca 2+The opening of channels into cells (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;

[0221] (2) Calcium entry induces depolarization and opening of voltage-dependent (L-VOCC) channels, which allows more Ca to enter. 2+ Entering cells (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. Feb. 2007; 100(4): 1089-96);

[0222] (3) This elevated intracellular calcium inducement includes an increase in the release of AChE G4 from T30 (Greenfield, 2013, “Discovering and targeting the basic mechanism of neurodegeneration: the role of peptides from the c-terminal of acetylcholinesterase Chemico-Biological Interactions”, 203(3): 543-6);

[0223] (4) Calcium also induces the upregulation of α7 nicotinic receptors, which will allow more calcium to be released. 2+By providing more targets for T30, it enters the cell (Bond et al., 2009, "Upregulation of alpha 7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides." Plos One, 4);

[0224] (5) Calcium-activating enzyme (i.e., GSK-3) will (a) increase τ protein, (b) activate γ-secretase / β-secretase, which will trigger the cleavage of extracellular toxic amyloid proteins, and (c) together with T30 will promote the release of even more toxic amounts of calcium. 2+ Entering the cell. (Hartigan and Johnson (1999), “Transient increase in intracellular calcium leads to increased site selectivity of prolonged τ protein phosphorylation via the β-dependent pathway of glycogen synthase kinase 3.” 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-Ratés et al. (2013), “Additive Toxicity of β-Amyloid by a Novel Bioactive Peptide InVitro: Possible Implications for Alzheimer's Disease,” PLoS ONE 8(2): e54864.) sequence list <110> NeuroBio Co., Ltd. <120> Animal models for neurodegenerative disorders <130> GBA1V220002443 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 614 <212> PRT <213> Homo sapiens <400> 1 Met Arg Pro Pro Gln Cys Leu Leu His Thr Pro Ser Leu Ala Ser Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Trp Leu Leu Gly Gly Gly Val Gly Ala Glu 20 25 30 Gly Arg Glu Asp Ala Glu Leu Leu Val Thr Val Arg Gly Gly Arg Leu 35 40 45 Arg Gly Ile Arg Leu Lys Thr Pro Gly Gly Pro Val Ser Ala Phe Leu 50 55 60 Gly Ile Pro Phe Ala Glu Pro Pro Met Gly Pro Arg Arg Phe Leu Pro 65 70 75 80 Pro Glu Pro Lys Gln Pro Trp Ser Gly Val Val Asp Ala Thr Thr Phe 85 90 95 Gln Ser Val Cys Tyr Gln Tyr Val Asp Thr Leu Tyr Pro Gly Phe Glu 100 105 110 Gly Thr Glu Met Trp Asn Pro Asn Arg Glu Leu Ser Glu Asp Cys Leu 115 120 125 Tyr Leu Asn Val Trp Thr Pro Tyr Pro Arg Pro Thr Ser Pro Thr Pro 130 135 140 Val Leu Val Trp Ile Tyr Gly Gly Gly Phe Tyr Ser Gly Ala Ser Ser 145 150 155 160 Leu Asp Val Tyr Asp Gly Arg Phe Leu Val Gln Ala Glu Arg Thr Val 165 170 175 Leu Val Ser Met Asn Tyr Arg Val Gly Ala Phe Gly Phe Leu Ala Leu 180 185 190 Pro Gly Ser Arg Glu Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Arg 195 200 205 Leu Ala Leu Gln Trp Val Gln Glu Asn Val Ala Ala Phe Gly Gly Asp 210 215 220 Pro Thr Ser Val Thr Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val 225 230 235 240 Gly Met His Leu Leu Ser Pro Pro Ser Arg Gly Leu Phe His Arg Ala 245 250 255 Val Leu Gln Ser Gly Ala Pro Asn Gly Pro Trp Ala Thr Val Gly Met 260 265 270 Gly Glu Ala Arg Arg Arg Ala Thr Gln Leu Ala His Leu Val Gly Cys 275 280 285 Pro Pro Gly Gly Thr Gly Gly Asn Asp Thr Glu Leu Val Ala Cys Leu 290 295 300 Arg Thr Arg Pro Ala Gln Val Leu Val Asn His Glu Trp His Val Leu 305 310 315 320 Pro Gln Glu Ser Val Phe Arg Phe Ser Phe Val Pro Val Val Asp Gly 325 330 335 Asp Phe Leu Ser Asp Thr Pro Glu Ala Leu Ile Asn Ala Gly Asp Phe 340 345 350 His Gly Leu Gln Val Leu Val Gly Val Val Lys Asp Glu Gly Ser Tyr 355 360 365 Phe Leu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp Asn Glu Ser Leu 370 375 380 Ile Ser Arg Ala Glu Phe Leu Ala Gly Val Arg Val Gly Val Pro Gln 385 390 395 400 Val Ser Asp Leu Ala Ala Glu Ala Val Val Leu His Tyr Thr Asp Trp 405 410 415 Leu His Pro Glu Asp Pro Ala Arg Leu Arg Glu Ala Leu Ser Asp Val 420 425 430 Val Gly Asp His Asn Val Val Cys Pro Val Ala Gln Leu Ala Gly Arg 435 440 445 Leu Ala Ala Gln Gly Ala Arg Val Tyr Ala Tyr Val Phe Glu His Arg 450 455 460 Ala Ser Thr Leu Ser Trp Pro Leu Trp Met Gly Val Pro His Gly Tyr 465 470 475 480 Glu Ile Glu Phe Ile Phe Gly Ile Pro Leu Asp Pro Ser Arg Asn Tyr 485 490 495 Thr Ala Glu Glu Lys Ile Phe Ala Gln Arg Leu Met Arg Tyr Trp Ala 500 505 510 Asn Phe Ala Arg Thr Gly Asp Pro Asn Glu Pro Arg Asp Pro Lys Ala 515 520 525 Pro Gln Trp Pro Pro Tyr Thr Ala Gly Ala Gln Gln Tyr Val Ser Leu 530 535 540 Asp Leu Arg Pro Leu Glu Val Arg Arg Gly Leu Arg Ala Gln Ala Cys 545 550 555 560 ​​​​​​​​​​​​​​​​​​​​​​​​​​Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 <210> 3 <211> 30 <212> PRT <213> Homo sapiens <400> 3 Lys Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys Asn 1 5 10 15 Gln Phe Asp His Tyr Ser Lys Gln Asp Arg Cys Ser Asp Leu​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

Claims

1. A method for providing an animal model for a neurodegenerative disease, the method comprising introducing a peptide into the brain of a non-human animal, said peptide comprising, or consisting of, an active variant of, an amino acid sequence represented as SEQ ID NO: 3 or a fragment thereof, wherein, The peptide causes an increase in τ protein at one or more sites in the brain of an animal; wherein the peptide or its variants or fragments comprise, or consist of, at least 25, 26, 27, 28 or 29 amino acids of the sequence represented by SEQ ID NO: 3; and wherein the concentration of the peptide or its variants or fragments administered to the animal is less than 75 µM, and the non-human animal is a rodent.

2. The method according to claim 1, wherein, (i) The method includes introducing a peptide or a variant or fragment thereof into the brain of a wild-type non-human animal; (ii) wherein administration of the peptide or its variants or fragments to the non-human animal causes an increase in τ protein at one or more sites in the animal’s brain, said sites being selected from the group consisting of: cortex, subcortex, hippocampus, cerebellum, basal forebrain, and pons and medullary regions; wherein administration of the peptide or its variants or fragments causes an increase in τ protein at at least one, two, three, four, five or all six sites in the animal’s brain, said sites being selected from the group consisting of: cortex, subcortex, hippocampus, cerebellum, basal forebrain, and pons and medullary regions; (iii) wherein, compared with an untreated control, administration of the peptide or its variants or fragments caused an increase of at least 1%, 3%, 5%, 10% or 20% of τ protein at one or more sites in the brain of an animal. (iv) wherein, compared with an untreated control, administration of the peptide or its variants or fragments caused an increase of at least 30%, 40% or 50% of τ protein at one or more sites in the brain of an animal. (v) wherein administration of the peptide or its variants or fragments to the non-human animal causes a reduction in neurons at one or more sites in the animal’s brain, said sites being selected from the group consisting of: cortex, subcortex, hippocampus, cerebellum, basal forebrain, and pons and medullary regions, wherein administration of the peptide or its variants or fragments causes an increase in τ protein at at least one, two, three, four, five, or all six sites in the animal’s brain, said sites being selected from the group consisting of: cortex, subcortex, hippocampus, cerebellum, basal forebrain, and pons and medullary regions.

3. The method according to claim 1 or 2, wherein, The peptide or its variants or fragments contain at least 25 or 30 amino acid residues, or consist of at least 15, 20, 25 or 30 amino acid residues, and have at least 90% or 95% sequence identity with SEQ ID NO:

3.

4. The method according to claim 1, wherein, (i) The concentration of the peptide or its variants or fragments administered to animals is less than 60 µM; (ii) wherein the concentration of the peptide or its variants or fragments 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; (iii) wherein the concentration of the applied peptide or its variant or fragment is greater than 0.01 µM, or greater than 0.1 µM, or greater than 1 µM, or greater than 3 µM, or greater than 5 µM, or greater than 10 µM, or greater than 20 µM; (iv) wherein the concentration of the applied peptide or its variant or fragment is greater than 30 µM, or greater than 40 µM, or greater than 50 µM, or greater than 60 µM, or greater than 70 µM; (v) wherein the concentration of the peptide or its variants or fragments is between 0.1 µM and 70 µM, or 0.1 µM and 60 µM, or 0.1 µM and 50 µM, or 0.1 µM and 40 µM, or 0.1 µM and 30 µM, or 0.1 µM and 20 µM, or between 0.1 µM and 10 µM; or (vi) wherein the concentration of the peptide or its variants or fragments is between 30 µM and 70 µM, or between 40 µM and 60 µM.

5. The method according to claim 1, wherein, Introducing the peptide, or its variants or fragments, into the basal forebrain region of the brain, and / or, The peptide or its variants or fragments are introduced into: (i) the septum / oblique band of the Broca region of the brain; (ii) the cholinergic system of the cortex; and / or (iii) the basal macrocell nucleus.

6. The method according to claim 1, wherein, (i) the rodent is a mouse or a rat; and / or (ii) the peptide or its variants or fragments contribute to or cause neurodegeneration.

7. The method according to claim 1, wherein, (i) Administration of the peptide or its variants or fragments to an animal model causes cell degeneration and thereby testable brain functional impairment, wherein the same brain functional impairment in humans represents a neurological disorder; (ii) wherein the method or model is used to study any neurodegenerative disease characterized by τ protein lesions; and / or (iii) wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease; Parkinson's disease; motor neuron disease; amyotrophic lateral sclerosis; Lewy body dementia and frontotemporal dementia; Among these, the experimentally tested brain function impairments that can be tested are cognitive or attention deficits.

8. The method according to claim 1, wherein, The method includes testing for appropriate brain function impairment in the animal model by providing the animal with an attention task to test for attention impairment; and / or, wherein the method further includes administering a test reagent before, simultaneously with, or after the peptide or its variants or fragments, and determining whether the test reagent inhibits, prevents, or increases testable brain function impairment and / or inhibits, prevents, or increases cell damage in the brain.

9. The method according to claim 8, wherein, Cellular damage includes neurodegeneration, wherein said cellular damage is monitored or assessed by measuring one or more of the following: (i) Inhibition of activity in a neuronal population; (ii) Calcium levels; (iii) The activity level of acetylcholinesterase; (iv) Expression of α-7 nicotinic receptors in the cell membrane; and (v) Cell density and / or loss of NeuN-expressing cells in a specific region.

10. An animal model for a neurodegenerative disease, comprising a non-human animal treated with a peptide, said peptide comprising an active variant of an amino acid sequence or fragment thereof represented as SEQ ID NO: 3, or consisting of an active variant of an amino acid sequence or fragment thereof represented as SEQ ID NO: 3; wherein, The peptide or its variants or fragments comprise at least 25, 26, 27, 28 or 29 amino acids of the sequence represented by SEQ ID NO: 3, or consist of at least 25, 26, 27, 28 or 29 amino acids of the sequence represented by SEQ ID NO: 3; and wherein the concentration of the peptide or its variants or fragments administered to an animal is less than 75 µM, and the non-human animal is a rodent.

11. The animal model according to claim 10, wherein, The animal model is prepared using the method according to any one of claims 1-9.

12. Use of the animal model according to claim 10 or 11 or the animal model prepared using the method according to any one of claims 1-9 for: (i) examining the process of neurodegeneration or neuroregeneration; (ii) testing a pharmacological compound that can modulate the process of neurodegeneration or neuroregeneration; or (iii) screening for drugs for neurodegeneration or neuroregeneration.

13. A method for identifying candidate reagents, said reagents being used to treat, prevent, or improve neurodegenerative disorders, said method comprising: - Administer the candidate reagent to an animal model according to claim 10 or 11 or prepared using the method according to any one of claims 1-9; and - To determine whether candidate reagents inhibit, prevent, or increase testable brain functional impairment and / or cause improvement or worsening of brain cell damage. Wherein, the inhibition or prevention of testable brain function impairment, or the improvement of brain cell damage, indicates that the test reagent is a candidate for the treatment, prevention, or improvement of neurodegenerative disorders, while the increase in testable brain function impairment or the deterioration of brain cell damage indicates that the test reagent is not a candidate for the treatment, prevention, or improvement of neurodegenerative disorders.

14. The method according to claim 13, wherein, The testable brain function impairment is a cognitive or attention deficit, wherein the method includes testing the animal model for impairment or cognitive or attention deficit.

15. A method for testing reagents used for biological activity, wherein, The method comprises administering the test reagent to an animal model according to claim 10 or 11, or an animal model prepared using the method according to any one of claims 1-9, and assessing any changes, improvements, or deteriorations in the brain injury-related aspects of the animal; wherein the assessment comprises determining whether the test reagent inhibits, prevents, or increases appropriate testable brain function impairment, cognitive function impairment, memory impairment, and / or determining whether there is any improvement or deterioration of cell damage at relevant sites in the brain; and wherein the test reagent is a pharmaceutical compound.

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