New synthetic drugs to treat Alzheimer's disease

Abstract

The present invention aims to provide novel drugs for treating Alzheimer's disease, methods for treating Alzheimer's disease, and methods for screening candidate substances for therapeutic drugs for Alzheimer's disease and the like. The present invention relates to a preventive and / or therapeutic agent for Alzheimer's disease, which comprises a peptide corresponding to dynamin 1. The peptide preferably corresponds to the dynamin 1-pleckstrin homology domain or the dynamin 1-proline-rich domain. Furthermore, the peptide is preferably encapsulated in nanoparticles or linked to a peptide sequence, which improves delivery of the peptide to the brain.

Description

[Technical Field]

[0001] The present invention relates to synthetic drugs for treating Alzheimer's disease, methods for treating Alzheimer's disease, and methods for screening candidate therapeutic agents for Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive dementia that begins with a decline in short-term memory and mild learning disabilities, and then develops into higher brain dysfunction, particularly visuospatial agnosia, ideational apraxia, and constructional apraxia, ultimately leading to motor disorders and personality disorders. To date, no method has been found that can completely cure Alzheimer's disease. It is predicted that the number of Alzheimer's disease patients worldwide will reach 2.4 million by 2040, and therefore new treatment methods are becoming increasingly important.

[0003] The microtubule (MT)-binding protein tau assembles and stabilizes MTs (1, 2), primarily in the axonal compartment (3, 4). Phosphorylation of tau protein reduces its binding affinity (5, 6), thereby shifting the equilibrium from MT-bound to a soluble, free form (7). Soluble tau protein also exists in a dynamic equilibrium between phosphorylated and dephosphorylated forms (8), as well as between soluble and aggregated forms. As cytoplasmic tau concentrations increase, monomeric tau undergoes oligomerization and ultimately deposits into neurofibrillary tangles (NFTs) (9–11). NFTs are a hallmark of tauopathies, including Alzheimer's disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP) (2, 7, 8). While NFT density can correlate with AD progression ( 2 , 7 , 12 ), soluble tau protein levels are more closely associated with disease progression and cognitive decline ( 13 , 14 ).

[0004] Genetic ablation of tau results in few abnormal phenotypes (15-17), likely due to compensation by other MT-associated proteins (15). Instead, tau ablation can prevent amyloid beta-induced impairment of mitochondrial transport (16) or memory impairment (18, 19). Thus, loss of tau function due to dissociation from MTs may not be a significant cause of neuronal dysfunction in AD (8, 12).

[0005] In postmortem brains of both AD patients and healthy individuals, tau is present in synaptosomes (20, 21). In transgenic mouse AD models, soluble tau accumulates in regions of hippocampal nerve terminals (22, 23). In both in vivo and culture models of tauopathy, upon KCl stimulation, tau acts like a neurotransmitter, transporting Ca2+. 2+ Tau is released from axon terminals in a synaptic-dependent manner (24, 25). Tau oligomers generated by the released tau induce endogenous tau propagation in neighboring neurons, thereby resulting in transsynaptic propagation (22, 26).

[0006] FTDP tauopathy mouse models overexpressing mutant tau have been widely used to examine the toxicity of tau on synaptic plasticity (27-29), memory formation (28, 30), and synaptic vesicle transport (31, 32). In contrast to FTDP, a rare familial disorder associated with tau mutations, AD is a widespread sporadic disease without tau mutations, but the expression level of wild-type tau is crucial. As an AD model, the effects of wild-type tau overexpression have been examined in cultured cells (33-36) or Drosophila (37), revealing impaired axonal transport associated with increased MT density. These observations suggest that elevated levels of wild-type tau may be deleterious (35, 36). However, unlike FTDP tau mutants, it is unclear whether elevated soluble wild-type tau can affect central synaptic transmission in mammals. [Prior art documents] [Non-patent literature]

[0007]

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[0008] An object of the present invention is to provide a novel drug for treating Alzheimer's disease, a method for treating Alzheimer's disease, and a method for screening candidate substances for therapeutic agents for Alzheimer's disease and the like. [Means for solving the problem]

[0009] We addressed the question of whether elevated soluble WT tau could affect central synaptic transmission in mammals using the calyx of Held, a large nerve terminal visualized in sections from mouse brainstem. In the calyx of Held, axonal MTs extend deep into the terminal (38). We modeled AD-associated elevated WT tau by introducing recombinant WT h-tau into this presynaptic terminal via a whole-cell patch pipette at a fixed concentration. We found that WT h-tau induces de novo assembly of MTs and strongly impairs synaptic transmission. Capacitance measurements indicated that the primary target of WT h-tau is vesicular endocytosis. Immunohistochemical imaging analysis after cell permeabilization revealed an increased MT-bound fraction of the endocytic GTPase dynamin in WT h-tau-introduced terminals. Because dynamin, a key endocytic protein, is a MT-binding protein (39), dynamin may be trapped by newly assembled MTs. Through screening, we found that a synthetic dodecapeptide corresponding to amino acids 560-571 of dynamin 1 inhibited MT-dynamin interaction. When we co-introduced this peptide, "PHDP5," with wild-type h-tau, its toxicity to vesicle endocytosis and synaptic transmission was rescued. Therefore, we propose a novel mechanism of synaptic dysfunction underlying AD, in which wild-type tau-induced overassembly of MTs depletes dynamin, thereby impairing vesicle endocytosis and synaptic transmission. Based on these novel findings, the present invention is as follows.

[0010] The present invention includes the following embodiments: (1) A preventive and / or therapeutic agent for Alzheimer's disease, comprising a peptide corresponding to dynamin 1. (2) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide corresponds to a dynamin 1-pleckstrin homology domain or a dynamin 1-proline-rich domain. (3) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, an amino acid sequence having one or more conservative amino acid substitutions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, or an amino acid sequence having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9. (4) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide is encapsulated in nanoparticles to improve delivery of the peptide to the brain. (5) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide is linked to a peptide sequence that improves delivery of the peptide to the brain. (6) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide sequence that improves delivery of the peptide to the brain is selected from the group consisting of SEQ ID NOs: 10 to 15. (7) The preventive and / or therapeutic agent for Alzheimer's disease according to (1), wherein the peptide is fused or conjugated to a compound that improves delivery of the peptide to the brain. (8) A preventive and / or therapeutic agent for Alzheimer's disease, comprising an inhibitor of microtubule-dynamin 1 binding. (9) The preventive and / or therapeutic agent for Alzheimer's disease according to (8), wherein the inhibitor is an isolated monoclonal antibody that binds to dynamin 1. (10) The preventive and / or therapeutic agent for Alzheimer's disease according to (8), wherein the inhibitor is an isolated monoclonal antibody that binds to the dynamin 1-pleckstrin homology domain or the dynamin 1-proline-rich domain. (11) An isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding, wherein the monoclonal antibody is an antibody against dynamin 1. (12) An isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding according to (11), wherein the monoclonal antibody is an antibody against the dynamin 1-pleckstrin homology domain or the dynamin 1-proline-rich domain. (13) An isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding according to (11), wherein the monoclonal antibody is an antibody against a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, an amino acid sequence having one or more conservative amino acid substitutions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, or an amino acid sequence having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9. (14) A method for screening for a substance effective in treating Alzheimer's disease, comprising measuring the activity of a test substance that inhibits the binding of microtubules and dynamin 1. [Effects of the Invention]

[0011] The present invention provides novel agents for treating Alzheimer's disease, methods for treating Alzheimer's disease, and methods for screening candidate substances for therapeutic agents for Alzheimer's disease and the like. [Brief explanation of the drawings]

[0012] [Figure 1]WT h-tau introduced into presynaptic terminals inhibited excitatory synaptic transmission. (A) Simultaneous presynaptic and postsynaptic whole-cell recordings showed that intraterminal injection of WT h-tau at 10 μM (blue circles) or 20 μM (black circles) through the tubing of a presynaptic patch pipette (top illustration) resulted in a concentration-dependent attenuation of EPSCs evoked by 1 Hz presynaptic action potentials. In the time plots, EPSC amplitudes averaged from 60 events are sampled as data points and normalized to the mean amplitude of baseline EPSCs before h-tau injection. Sample recordings of EPSCs 5 min before (i) and 30 min after (ii) tau injection are overlaid and shown in the left panel. Thirty minutes after injection, the remaining EPSC amplitudes were 23 ± 9% and 65 ± 5% for 10 μM and 20 μM h-tau, respectively (mean and SEM, six synapses from six slices, p < 0.01 by paired t-test between before and after h-tau injection). Injection of the MT-binding site-deleted h-tau mutant (del-MTBD, 20 μM, Figure 10A) did not affect EPSC amplitude (open circles, sample EPSC trace shown in the bottom left panel). (B) The amplitude of EPSCs evoked at 0.1 Hz remained unchanged after h-tau injection (85% ± 12%, five synapses from five slices, p = 0.22 by paired t-test). Sample recordings of EPSCs before (i) and 30 minutes after (ii) h-tau injection at 0.1 Hz are overlaid in the left panel. (C) Taxol (1 μM) produced an activity-dependent attenuation of EPSC amplitude by 41.4 ± 12% at 1 Hz (p < 0.01, 5 synapses from 5 slices) but remained unchanged when stimulated at 0.1 Hz (105 ± 3.0%, open circles, 4 synapses from 4 slices). Sample EPSCS recordings at 0.1 Hz and 1 Hz are overlaid in the left panel. [Figure 2]Figure 1 shows that inhibition of SV endocytosis is the primary effect of WT h-tau introduction. (A) Exo-endocytic membrane capacitance changes in presynaptic terminals after direct introduction of WT h-tau (20 μM; tau) without (control) or after direct introduction. WT h-tau was introduced directly into the terminal by diffusion from a whole-cell patch pipette (illustration). Capacitance traces were sampled (overlaid) at (i) 10 min, (ii) 20 min, and (iii) 30 min after patch membrane disruption. Left panel: unintroduced control. Right panel: WT h-tau-introduced terminal. Capacitance changes were elicited every 2 min by Ca2+ currents induced by a 20 ms depolarizing pulse (not shown). (B) Time plots of endocytosis rate (left panel), extent of exocytosis (middle panel), and presynaptic Ca2+ current charge (right panel). Data points represent averages from five events 4 min before and 4 min after the time point. At the calyceal terminal, 20 min after patch membrane disruption with a pipette containing WT h-tau (filled circles; tau), the endocytosis rate was significantly prolonged (p < 0.05 compared with control; open circles; repeated-measures two-way ANOVA with post-hoc Scheffe's test; n = 5 from 5 sections), whereas the extent of exocytosis remained similar to control (p = 0.45). Thirty min after disruption, the endocytosis rate was further prolonged (p < 0.01), and the extent of exocytosis was significantly lower than control (p < 0.05). The Ca2+ current charge (QCa) remained unchanged throughout the recording. [Figure 3]This figure shows that the MT assembly inhibitor nocodazole prevented tau-induced inhibition of SV endocytosis and EPSC attenuation. (A) Concentration-dependent inhibitory effect of nocodazole on MT assembly in a tubulin polymerization assay. MT assembly by 0N4R h-tau (20 μM) in the absence (pink symbols and fitting line) or presence of nocodazole at 1 μM (blue), 10 μM (red), 20 μM (purple), and 50 μM (orange) concentrations. Data points and error bars in all graphs represent the mean and SEM (n = 3). (B) Nocodazole prevented h-tau-induced inhibition of SV endocytosis. Presynaptic membrane capacitance changes (overlaid traces) for 25 min with h-tau alone (20 μM, red trace), h-tau and nocodazole (20 μM, blue), nocodazole alone (20 μM, green), and no control (black). Bar graphs show endocytosis rates for untransfected control (Ctr, black, 6 terminals from 6 sections), h-tau-transfected terminals (Tau, red, 8 terminals from 8 sections), nocodazole and h-tau cotransfection (N+T, blue, 7 terminals from 7 sections), and nocodazole alone (Noc, green, 8 terminals from 8 sections). Nocodazole cotransfection completely prevented h-tau-induced endocytosis inhibition compared to control levels (p<0.01, between Tau and NT) (one-way ANOVA with Scheffe post-hoc test). (C) Nocodazole prevented EPSC attenuation caused by WT h-tau. Nocodazole (20 μM) cotransfected with WT h-tau (20 μM) prevented EPSC attenuation (black circles, 4 synapses from 4 sections; p<0.01, unpaired t-test). Data for the effect of WT h-tau on EPSCs (Fig. 1A) are shown as dashed red lines for comparison. Nocodazole alone (20 μM) had no effect on EPSC amplitude throughout (open circles, four synapses from four slices). [Figure 4]Increased WT h-tau MT assembly and bound dynamin at calyx terminals. (A) Immunofluorescence images of brainstem sections showing WT h-tau transfection (green, left panel, arrowheads) labeled with anti-h-tau / AlexaFluor-488 antibody, newly assembled MTs labeled with anti-β3 tubulin / AlexaFluor-647 antibody (magenta, middle panel), and increased bound dynamin labeled with anti-dynamin 1 / AlexaFluor-568 antibody (red, right panel). (B) Bar graphs showing immunofluorescence intensity of h-tau (green), β3 tubulin (magenta), and dynamin (red) compared to untransfected controls (black bars). WT h-tau transfection significantly increased terminal β3-tubulin (p=0.0105) and dynamin 1 (p=0.0109) intensities compared to control terminals without WT h-tau transfection (n=5 terminals from 5 sections for each data set, unpaired two-tailed t-test with Welch's correction; *p<0.05, ***p<0.001). [Figure 5]Dynamin 1 PH domain peptides inhibited MT-dynamin 1 binding and prevented the slowing of endocytosis and EPSC attenuation caused by WT h-tau. (A) Top: Partial amino acid sequence of the PH domain of mouse dynamin 1 showing the sequence (560-571) of the synthetic dodecapeptide PHDP5. Left: SDS-PAGE of the MT-dynamin 1 binding assay. S, supernatant; P, precipitate. Dyn1, dynamin 1. Right: Quantification of MT-dynamin 1 interaction. Bars indicate the percentage of dynamin 1 found in the precipitate relative to the total amount. PHDP5 significantly inhibited MT-dynamin 1 interaction (**<0.01, n=3). (B) Presynaptic membrane capacitance recordings (overlaid) after transfection with h-tau alone (20 μM, red trace, excerpt from Figure 4B), h-tau cotransfected with DPHP5 (0.25 mM, blue), or scrambled DPHP5 (SDPHP5, green). DPHP5 alone (0.25 mM, black trace, seven terminals from seven sections) did not affect capacitance changes compared to the untransfected terminal control (excerpt from Figure 3B). Bar graphs of endocytosis rates (middle panel) show significant differences between tau (red bars, eight terminals from eight sections) and DPHP5 + tau (blue, seven terminals from seven sections), and between SDPHP5 + tau (blue) and DPHP5 + tau (green, terminals from six sections, n = 6) (p < 0.05, seven terminals from seven sections). The magnitude of exocytic capacitance changes recorded 25 min after the lesion did not differ significantly between groups. (C) DPHP5 attenuated h-tau-induced EPSC attenuation. EPSC attenuation after h-tau injection (20 μM, red dashed line; data extracted from Figure 1A) was attenuated by DPHP5 (1 mM) co-injected with h-tau (filled circles), but not by scrambled DPHP5 peptide (SDPHP5, open triangles, 1 mM). DPHP5 alone (1 mM, open circles) had no effect on EPSC amplitude throughout. Bar graphs show EPSC amplitudes 30 min after injection (normalized to pre-injection EPSC amplitude). Significant differences (p<0.01) were observed between tau and tau + DPHP5, and between tau + DPHP5 and tau + SDPHP5.The difference between DPHP5 alone and DPHP5 plus tau was not significant (p=0.09), indicating a partial antagonism of DPHP5 on h-tau-induced EPSC attenuation. [Figure 6A] First, MTs were prepared from tubulin and stabilized with taxol. Then, taxol-stabilized MTs and Dyn1 were incubated with or without PHDP5. Supernatant (unbound protein) and pellet (bound protein) fractions were separated using ultracentrifugation. Samples were analyzed by SDS-PAGE gel and visualized by SYPRO Orange staining. The results showed that the percentage of Dyn1 in the pellet fraction did not differ between the supernatant and pellet fractions after incubation with PHDP5. This may be due to competitive binding of Dyn1 / PHDP5 to MTs. [Figure 6B] We changed the incubation order by mixing MT with PHDP5 and then incubating with Dyn1, and the results showed that the percentage of Dyn1 was decreased in the pellet fraction in the presence of PHDP5. [Figure 7A] The results showed that the percentage of Dyn1 in the pellet fraction was lower when MTs were incubated with 3 mM PHDP5 than when they were incubated with 1 mM PHDP5, revealing its potential to inhibit MT-dynamin interaction. [Figure 7B] When MTs were incubated with 1 mM PHDP5, shorter incubation periods with Dyn1 (10 min) resulted in a lower percentage of Dyn1 in the pellet fraction. Without PHDP5, there was no difference between the time points. [Figure 7C] When MTs were incubated with PHDP5 3 mM, a 5 min incubation with Dyn1 showed a lower % of Dyn1 in the pellet fraction. [Figure 7D] Under the same conditions, several FITC-labeled PHDP5 candidates and their scrambled controls were also tested. The first candidate showed a lower % of Dyn1 than PHDP5. However, more testing is needed in our laboratory. [Figure 8A]This figure shows the statistical results of Figure S4. [Figure 8B] This figure shows the statistical results of Figure S4. [Figure 9] Electron images of negatively stained microtubules and dynamin 1 in the presence or absence of PHDP5. MT-dynamin 1+FP5#3 appears to stabilize the MT-dynamin 1 interaction. [Figure 10] Figure 1 shows tubulin polymerization assays of purified 0N4R WT h-tau and its MT-binding domain deletion mutant. (A) Left panel: Schematic diagram of WT 0N4R h-tau (a) and the h-tau deletion mutant lacking the MT-binding domain (del-MTBD, b). Right panel: SDS-PAGE of purified recombinant WT 0N4R h-tau (a) and del-MTBD (b) with molecular markers (M) in the left lane. (B) In vitro tubulin polymerization assay showing MT assembly by WT h-tau (10 μM, open circles) or Taxol (1 μM, filled triangles), but not by del-MTBD (10 μM, open squares) or tubulin alone (filled circles). Data points and bars represent the mean and SEM (n = 3). [Figure 11] Figure 1 shows that PHDP5 strongly inhibits dynamin 1 binding to microtubules. (A) Peptide sequence of mouse dynamin 1 used in the DNA peptide screening. A total of 22 peptides covering the PH domain and proline-rich domain were synthesized. (B) SDS-PAGE of the microtubule-binding assay (upper right panel, "+Pep5") showing that PHDP5 strongly inhibits MT-dynamin 1 binding. Five peptides (peptide 6, peptide 8, peptide 9, peptide 10, and peptide 11) were insoluble in water or dimethyl sulfoxide, so their effects were not tested. (C) Effect of 1 mM synthetic peptide on MT-dynamin 1 binding. The bar graph shows the percentage of dynamin 1 found in the precipitate relative to the total amount. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise specified, all terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context dictates otherwise. Unless otherwise specified, molecular biological techniques used herein can be performed by methods described in general laboratory manuals known to those skilled in the art, or methods similar thereto.

[0014] As used herein, "corresponding to" or "corresponds to" means that the preceding term corresponds to a part or all of a molecule, domain, etc. For example, "a peptide corresponding to dynamin 1" means that the peptide corresponds to a part or all of dynamin 1. The peptide may or may not be completely identical to dynamin 1, but may be a peptide having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity.

[0015] The present inventors have demonstrated that tau, a microtubule-binding protein implicated in Alzheimer's disease, reduces synaptic signaling in mouse brains. Tau protein, produced in neurons, binds to microtubules and participates in microtubule assembly. Normally, tau protein is either bound to microtubules or dissolved in the fluid inside the cell. However, in some neurological disorders, particularly Alzheimer's disease, excessive concentrations of soluble tau protein occur in certain brain regions, where it aggregates to form insoluble structures called neurofibrillary tangles. The present inventors investigated the effect of high concentrations of soluble tau protein on signal transduction at the calyx of Held, the largest synapse in the mammalian brain. Injection of soluble tau protein into the presynaptic terminal of mouse Held calyx synapses significantly reduced electrical signal generation in postsynaptic cells. Fluorescent labeling of tau protein and microtubules confirmed that tau injection induced the assembly of numerous new microtubules at the presynaptic terminal. However, when they instead injected a mutant tau protein lacking the binding site involved in microtubule polymerization, synaptic signaling was unaffected.

[0016] We found that increased tau protein only reduced the transmission of high-frequency signals, leaving low-frequency signal transmission unchanged. Because high-frequency signals are commonly involved in cognition and motor control, we investigated the possibility that increased tau protein inhibits synaptic vesicle recycling. We found that increasing the concentration of soluble tau protein initially inhibited endocytosis. Furthermore, fluorescent labeling of tau protein, microtubules, and dynamin-1 revealed that tau-injected presynaptic terminals exhibited increased dynamin-1 binding, preventing it from playing a role in endocytosis. Therefore, we synthesized a number of peptides with amino acid sequences partially identical to dynamin-1 and investigated whether these peptides could prevent tau signaling by inhibiting dynamin-1 binding to microtubules. Among these peptides, we found that a peptide containing a partial sequence of the pleckstrin homology domain of dynamin-1 prevented signaling disruption and maintained nearly normal endocytosis and signaling.

[0017] Based on these new findings, the present invention provides a therapeutic agent for Alzheimer's disease, which comprises a peptide that inhibits the binding of dynamin-1 protein to microtubules, a method for treating Alzheimer's disease, which comprises administering a peptide that inhibits the binding of dynamin-1 protein to microtubules, and a method for screening for substances effective in treating Alzheimer's disease. The present invention is described in detail below.

[0018] <Treatment for Alzheimer's disease> The therapeutic agent for Alzheimer's disease contains a peptide that inhibits the binding of dynamin-1 protein to microtubules. Furthermore, the therapeutic agent for Alzheimer's disease may contain, in addition to the above-mentioned peptide, other components to the extent that the effects of the present invention are not impaired. The therapeutic agent for Alzheimer's disease of the present invention is described in detail below.

[0019] (peptide) Dynamin is a GTPase involved in eukaryotic endocytosis and a member of the dynamin protein family. Dynamin family proteins play a role in many processes, including scission of newly formed vesicles from the plasma membrane or Golgi membrane, organelle division, cytokinesis, and microbial pathogen resistance. Three distinct dynamins have been identified in mammals, with significant amino acid sequence differences in their pleckstrin homology domains. One of them, dynamin 1, is expressed in neurons and neuroendocrine cells (Nature review Molecular cell biology, 2012, 13:75-88).

[0020] The peptides contained in the therapeutic agent for Alzheimer's disease that inhibit the binding of dynamin-1 protein to microtubules are not particularly limited as long as they inhibit the binding of dynamin-1 protein to microtubules. Examples include proteins corresponding to dynamin 1 protein (human: SEQ ID NO: 1, mouse: SEQ ID NO: 2), i.e., a portion or the entire dynamin 1 protein (which may or may not be completely identical to a portion or the entire dynamin 1 protein, e.g., having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity). Among these, peptides containing the entire or partial amino acid sequences of the pleckstrin homology domain of dynamin 1 (human: SEQ ID NO: 3, mouse: SEQ ID NO: 4) and the proline-rich domain of dynamin 1 (human: SEQ ID NO: 5, mouse: SEQ ID NO: 6) are preferred, peptides containing the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are more preferred, and peptides containing the amino acid sequence of SEQ ID NO: 7 are even more preferred. The amino acid sequences of the pleckstrin homology domain and the proline-rich domain of dynamin 1 are the same in humans and mice.

[0021] In addition to peptides containing the above amino acid sequences, peptides containing the above amino acid sequences with one or more conservative amino acid substitutions are also preferred as peptides to be contained in the therapeutic agents for Alzheimer's disease of the present invention. The above conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0022] In addition to peptides containing the above amino acid sequences, peptides having at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, and even more preferably at least 99% amino acid sequence identity with the above amino acid sequences are also listed as peptides contained in therapeutic agents for Alzheimer's disease.

[0023] The term "sequence identity" refers to the amount (number) of amino acid sequences (or polypeptide sequences) or polynucleotide sequences (or nucleotide sequences) between two chains that can be determined to be identical in terms of the correspondence between each amino acid residue or each nucleotide that makes up the chains. The term refers to the degree of sequence correlation between two amino acid sequences or two polynucleotide sequences. Identity can be readily calculated. Many methods are known for measuring identity between two amino acid sequences or polynucleotide sequences, and the term "sequence identity" is well known to those skilled in the art.

[0024] Furthermore, the peptides contained in the therapeutic agents for Alzheimer's disease of the present invention include peptides that contain the above amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added, and have the function of inhibiting the binding of microtubules to dynamin 1 protein. Here, "several" may be at most 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids.

[0025] Mutant DNAs can be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, and mutagenesis. Specifically, mutant DNAs can be obtained by introducing mutations into DNA consisting of the nucleotide sequences set forth in the SEQ ID NOs. encoding the above amino acid sequences using contact interaction with mutagens, ultraviolet irradiation, genetic engineering, or other methods. Site-directed mutagenesis, a genetic engineering method, is useful here. This is because, as described in Sambrook, J. et al., Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, site-directed mutagenesis can introduce specific mutations at specific positions. By expressing mutant DNAs using an appropriate expression system, peptides containing amino acid sequences in which one or more amino acids have been deleted, substituted, inserted, or added can be obtained.

[0026] The peptide contained in the therapeutic agent for Alzheimer's disease of the present invention may contain additional amino acid residues in the above amino acid sequence, as long as the peptide exhibits the effect of inhibiting the binding of dynamin-1 protein to microtubules. For example, the N-terminus and / or C-terminus of the above amino acid sequence may contain at least one additional amino acid residue.

[0027] The additional amino acid residues are added for, for example, production, purification, and stabilization of the polypeptide in vivo, binding to other molecules, and detection. The additional amino acid residues may also be a polypeptide having brain transport activity, an amino acid motif sequence, etc.

[0028] The peptide contained in the therapeutic agent for Alzheimer's disease may be all L-amino acids, all D-amino acids, or a mixture of L-amino acids and D-amino acids, but a peptide containing all L-amino acids is preferred. Peptides containing two or more asymmetric carbon atoms can be in any form of enantiomers or diastereomers in any ratio.

[0029] The length of the peptide contained in the therapeutic agent for Alzheimer's disease of the present invention is not particularly limited, but is preferably 5 to 50 amino acid residues, more preferably 8 to 40 amino acid residues, and most preferably 10 to 35 amino acid residues.

[0030] The peptide contained in the therapeutic agent for Alzheimer's disease of the present invention can be obtained according to a method known in the art, for example, by liquid phase peptide synthesis or solid phase peptide synthesis.

[0031] The peptides contained in the therapeutic agents for Alzheimer's disease of the present invention may be in the form of salts, preferably containing pharmaceutically acceptable counterions (e.g., chloride, sulfate, citrate, phosphate, acetate, sodium, potassium, calcium, magnesium). The peptide salts may be acid addition salts or base addition salts. Exemplary acids that can be used to form acid addition salts include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Exemplary bases that can be used to form base addition salts include sodium hydroxide, potassium hydroxide, alkali metal bases such as lithium hydroxide and calcium hydroxide, and alkaline earth metal bases such as magnesium hydroxide.

[0032] The peptide contained in the therapeutic agent for Alzheimer's disease may have some or all of the amino acid residues in the amino acid sequence modified. Such modified peptides can be prepared by any method known in the art. For example, modified peptides can be prepared by modifying the functional groups of the side chains of the amino acid residues that constitute the peptide, such as esterification, alkylation, halogenation, phosphorylation, sulfonation, amidation, etc.

[0033] The peptide contained in the therapeutic agent for Alzheimer's disease may be modified to increase its permeability through the blood-brain barrier.

[0034] Peptides contained in Alzheimer's disease therapeutic agents can be supplemented with polypeptides, amino acid motif sequences, and the like to enhance membrane permeability and promote brain delivery. Non-limiting examples of polypeptides and amino acid motif sequences that can be supplemented are described in, for example, WO1989010134, WO2005014625, WO2019126240, and US Pat. No. 7,927,811, all of which are incorporated herein by reference. Specific examples of polypeptides and amino acid motif sequences that can be supplemented with peptides contained in Alzheimer's disease therapeutic agents to enhance membrane permeability and promote brain delivery include peptides containing the sequences set forth in SEQ ID NOS: 10 to 15 (Table 1), and peptides to which these polypeptides have been supplemented that are contained in Alzheimer's disease therapeutic agents include peptides set forth in SEQ ID NOS: 16 to 27 (Table 2).

[0035] [Table 1]

[0036] [Table 2]

[0037] A peptide contained in a therapeutic agent for Alzheimer's disease can be modified, for example and without limitation, by adding, fusing, or binding to a receptor expressed in the blood-brain barrier (non-limiting examples include an antigen-binding fragment derived from an antibody that binds to an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, a lipoprotein receptor, or a transferrin receptor), thereby increasing membrane permeability and promoting transfer into the brain.

[0038] The peptides contained in the Alzheimer's therapeutic agents of the present invention can also be introduced into nanocapsules made of nanoparticles that can cross the blood-brain barrier (a non-limiting example is that described in Prog Neuropsychopharmacol Biol Psychiatry, 23, pp. 941-949, 1999, the entire contents of which are incorporated herein by reference) to facilitate their transport to the brain.

[0039] The peptides contained in the therapeutic agents for Alzheimer's disease of the present invention may be fused, bound, or attached with other substances for the purpose of improving their blood half-life, etc. The peptides may be bound or linked to specific substances at the N-terminus and / or C-terminus of the peptide via chemicals such as cross-linking agents, via drugs suitable for linking to amino acid side chains, or by synthetic chemical methods or genetic engineering methods. Examples of such substances include polyalkylene glycol molecules such as polyethylene glycol (PEG); fatty acid molecules such as hydroxyethyl starch and palmitic acid; Fc regions of immunoglobulins; CH3 domains of immunoglobulins; CH4 domains of immunoglobulins; albumin or fragments thereof; albumin-binding peptides; albumin-binding proteins such as streptococcal protein G; and transferrin. These substances are appropriately used to adjust the solubility of peptides, improve peptide stability by improving protease resistance, etc., and deliver peptides to specific tissues or organs.

[0040] The therapeutic agent for Alzheimer's disease of the present invention can contain the above-mentioned peptide in any appropriate amount, specifically, 0.1 to 100 wt%, 1 to 99 wt%, 1 to 90 wt%, 5 to 80 wt%, 10 to 75 wt%, or 15 to 50 wt% of the therapeutic agent for Alzheimer's disease.

[0041] Other ingredients In addition to the peptide, the therapeutic agent for Alzheimer's disease of the present invention may contain a pharmaceutically acceptable carrier, diluent, and / or excipient. Specifically, for example, if the therapeutic agent for Alzheimer's disease of the present invention is a liquid composition, the therapeutic agent may contain a suitable solvent such as water, physiological saline, glucose solution, or ethanol. The therapeutic agent for Alzheimer's disease of the present invention can be formulated, for example, by dissolving or suspending a fixed amount of the peptide in a solvent. A buffer, a preservative, a flavoring, and / or a coloring agent may each be included in the formulation as needed. When the therapeutic agent for Alzheimer's disease of the present invention is a separate composition, the composition may contain a binder, a lubricant, a disintegrant, a coloring agent, a flavoring agent, a glidant, and / or a solubilizer. For example, a solid formulation may contain an inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, crystalline cellulose, etc. Examples of binders include sugars such as starch, gelatin, glucose, lactose, and trehalose, natural and synthetic gums such as corn starch, calcium lactate, acacia, tragacanth, and sodium alginate, povidone, carboxymethylcellulose, hydroxypropylcellulose, polyethylene glycol, and waxes. Examples of lubricants include sodium oleate, sodium stearate, magnesium stearate, stearic acid, sodium stearyl fumarate, anhydrous silicic acid, talc, etc. Examples of disintegrants include starch, methylcellulose, agar, bentonite, xanthan gum, croscarmellose sodium, sodium starch glycolate, etc. Solid formulations may be enteric coated using, for example, methyl methacrylate polymers, ethyl cellulose, or carnauba wax.

[0042] The therapeutic agent for Alzheimer's disease of the present invention can be formulated for oral or parenteral (e.g., intramuscular or intravenous) administration. The method of administration may vary depending on the condition, age, etc. of the subject. The therapeutic agent for Alzheimer's disease of the present invention can be formulated into any appropriate dosage form such as tablets, injection solutions, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, etc., by techniques known in the art.

[0043] An appropriate daily dose of the therapeutic agent for Alzheimer's disease of the present invention may be in the range of 0.005 mg to 500 mg per kg of body weight per day as the amount of the above-mentioned peptide (e.g., 0.005 mg / kg to 100 mg / kg, 0.005 mg / kg to 30 mg / kg, 0.005 mg / kg to 1 mg / kg, 0.01 mg / kg to 30 mg / kg, 0.01 mg / kg to 3 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.02 mg / kg to 5 mg / kg, 0.02 mg / kg to 2 mg / kg, or 0.02 mg / kg to 1 mg / kg).

[0044] <Therapeutic agent for Alzheimer's disease containing a dynamin 1-microtubule binding inhibitor> The present invention includes therapeutic agents for Alzheimer's disease containing inhibitors of the binding between dynamin 1 protein and microtubules. Based on the mechanism elucidated above, the binding inhibitors are not limited to those that inhibit the binding between dynamin 1 protein and microtubules, and include not only the specific peptides described above, but also low-molecular-weight compounds, medium-molecular-weight compounds (e.g., peptides other than those described above), and high-molecular-weight compounds (e.g., antibodies).

[0045] Monoclonal antibodies against dynamin 1 protein are particularly effective as the binding inhibitor. Non-limiting examples include monoclonal antibodies against proteins corresponding to dynamin 1 protein (human: SEQ ID NO: 1, mouse: SEQ ID NO: 2), i.e., against a portion or the entire dynamin 1 protein. The antibody may or may not be completely identical to a portion or the entire dynamin 1 protein; for example, antibodies with at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity are useful. Among these, monoclonal antibodies against the pleckstrin homology domain of dynamin 1 (human: SEQ ID NO: 3, mouse: SEQ ID NO: 4), the proline-rich domain of dynamin 1 (human: SEQ ID NO: 5, mouse: SEQ ID NO: 6), and peptides containing all or part of the amino acid sequences of SEQ ID NOs: 7, 8, and 9 are preferred. Monoclonal antibodies against peptides containing the amino acid sequences of SEQ ID NOs: 7, 8, and 9 are even more preferred.

[0046] The antibodies used in the present invention may be derived from any source, and are not particularly limited, and may include antibodies preferably derived from mammals, more preferably from humans. Monoclonal antibodies derived from mammals include antibodies produced by hybridomas and antibodies produced by hosts transformed using genetic engineering techniques with expression vectors containing antibody genes.

[0047] Antibody-producing hybridomas can be produced essentially using known techniques, as described below. More specifically, dynamin 1 is used as a sensitizing antigen for immunization according to conventional immunization methods. The resulting immunocytes are fused with known parent cells according to conventional cell fusion methods. The fused cells are screened for monoclonal antibody-producing cells according to conventional screening methods.

[0048] Specifically, anti-dynamin 1 antibodies can be produced as follows: For example, human dynamin 1, which is used as an antigen to obtain the antibodies, can be obtained using the dynamin 1 gene / amino acid sequence disclosed in known literature.

[0049] The sequence of the dynamin 1 gene is inserted into any known expression vector system. The resulting expression vector system is used to transform an appropriate host cell. The desired dynamin 1 protein is then purified from the host cell or the culture supernatant of the host cell using any known method. The purified dynamin 1 protein can be used as a sensitizing antigen. Dynamin 1 protein produced by chemical synthesis can also be used as a sensitizing antigen. A fusion protein between dynamin 1 protein and any other protein can also be used as a sensitizing antigen.

[0050] The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. Typically, rodents such as mice, rats, and hamsters are used.

[0051] Immunization of animals with a sensitizing antigen is carried out according to any known method. For example, immunization is generally carried out by intraperitoneally or subcutaneously injecting the sensitizing antigen into a mammal. Specifically, the sensitizing antigen is diluted with phosphate-buffered saline (PBS), saline, or the like to obtain an appropriate volume of suspension. The suspension is optionally mixed with an appropriate amount of a conventional adjuvant, such as complete Freund's adjuvant, and then emulsified. The emulsified product is preferably administered to the mammal in a certain dose every 4 to 21 days. A suitable carrier can also be used for immunization with the sensitizing antigen.

[0052] After immunization and confirming an increase in the level of the desired antibody in the serum, immune cells are removed from the mammal and subjected to cell fusion. Preferred immune cells for cell fusion include spleen cells in particular.

[0053] The immune cells are fused with other parent cells. Suitable parent cells include mammalian myeloma cells, including a variety of known cell lines.

[0054] Cell fusion between immune cells and myeloma cells can be carried out essentially using any known method, for example, according to the method of Milstein et al. (Methods Enzymol., 73, pp. 3-46 (1981)).

[0055] More specifically, cell fusion is carried out in a standard nutrient medium in the presence of a cell fusion promoter. Cell fusion promoters that can be used include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). Furthermore, to enhance fusion efficiency, auxiliary agents such as dimethyl sulfoxide may be added as desired.

[0056] The immune cells and myeloma cells are preferably used in a ratio such that the number of immune cells exceeds the number of myeloma cells by 1 to 10 times. Media that can be used in cell fusion include, for example, RPMI1640 medium and MEM medium (both suitable for the growth of myeloma cell lines), as well as other conventional media used in this type of cell culture. Furthermore, serum complements such as fetal calf serum (FCS) can also be used.

[0057] In cell fusion, a given amount of immune cells and myeloma cells are thoroughly mixed in a medium. Next, a PEG solution preheated to 37°C, typically at a concentration of 30-60% (w / v), with an average molecular weight of approximately 1000-6000, is added and mixed to form the desired fused cells (hybridomas). To remove cell fusion agents and other agents that may be unfavorable to hybridoma growth, the following steps may be repeated sequentially: adding an appropriate medium, centrifuging the resulting suspension, and removing the supernatant.

[0058] Hybridomas are selected by culturing them in a conventional selective medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culturing in HAT medium is continued for a period sufficient to kill any cells other than the desired hybridoma (unfused cells), usually several days to several weeks. Hybridomas producing the desired antibody are then screened and cloned using conventional limiting dilution methods.

[0059] Methods for obtaining monoclonal antibodies from hybridomas include culturing hybridomas according to any conventional method and recovering the culture supernatant to obtain the monoclonal antibody, or administering the hybridomas to a mammal compatible with the hybridomas, allowing the hybridomas to grow, and recovering the ascites to obtain the monoclonal antibody. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

[0060] The monoclonal antibody used in the present invention may be a recombinant antibody produced by genetic engineering techniques, which involve cloning an antibody gene from a hybridoma, inserting the gene into an appropriate vector, and introducing the vector into a host (see, for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, 1990, published in the UK by MACMILLAN PUBLISHERS LTD).

[0061] Specifically, mRNA encoding the variable (V) region of the antibody of interest is isolated from antibody-producing cells, such as hybridomas. The mRNA is isolated by preparing total RNA using any known method, such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, pp. 5294-5299) or the AGPC method (Anal. Biochem. (1987) 162, pp. 156-159), and then preparing mRNA using, for example, an mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared using a QuickPrep mRNA Purification Kit (Pharmacia).

[0062] The resulting mRNA is used with reverse transcriptase to synthesize cDNA for the antibody V region. cDNA can be synthesized using the AMY Reverse Transcriptase First-Strand cDNA Synthesis Kit or similar. cDNA can also be synthesized and amplified using 5'-RACE (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acid Res. (1989) 17, 2919-2932) and PCR using the 5'-Ampli FINDER RACE Kit (Clontech). The resulting PCR product is purified to obtain the desired DNA fragment, which is then ligated to vector DNA. The desired recombinant vector can then be prepared by using the ligated vector DNA to create a recombinant vector, introducing the recombinant vector into Escherichia coli cells, or the like, and selecting colonies. The nucleotide sequence of the desired DNA can be confirmed using any known method, such as the deoxyribonucleotide method.

[0063] Once DNA encoding the V region of the antibody of interest is obtained, this DNA is ligated to DNA encoding the desired antibody constant region (C region), and the ligated DNA is inserted into an expression vector. Alternatively, DNA encoding the antibody V region may be inserted into an expression vector containing DNA for the antibody C region.

[0064] To produce the antibodies used in the present invention, the antibody genes are inserted into an expression vector to express the antibody genes under the control of expression regulatory regions such as enhancers and promoters described below. The expression vector is then transformed into a host cell that allows for the expression of the antibody.

[0065] Genetically modified recombinant antibodies that have been artificially modified to reduce heterologous antigenicity to humans and the like can be used in the present invention. Genetically modified antibodies include, for example, chimeric antibodies, humanized antibodies, and human antibodies. These modified antibodies can be produced using any known method.

[0066] A chimeric antibody can be obtained by ligating DNA encoding the V region of the antibody obtained as described above to DNA encoding the C region of a human antibody, inserting the ligated DNA into an expression vector, introducing the expression vector into a host, and allowing the host to produce the chimeric antibody (see European Patent Application Publication No. EP125023 and International Publication No. WO92-19759). This known procedure can be used to obtain chimeric antibodies useful in the present invention.

[0067] Humanized antibodies, also called reshaped human antibodies or human-type antibodies, are antibodies in which the complementarity-determining regions (CDRs) of an antibody derived from a non-human mammal, such as a mouse, are grafted onto the complementarity-determining regions of a human antibody. General genetic recombination techniques for producing humanized antibodies are known (see European Patent Application Publication No. EP125023 and International Publication No. WO92-19759).

[0068] Specifically, a DNA sequence designed to link the CDR of a mouse antibody to the framework region (FR) of a human antibody is synthesized using PCR with several oligonucleotides that are generated so that the ends of the oligonucleotides have overlapping sequences. The resulting DNA is ligated to the DNA encoding the C region of a human antibody and then inserted into an expression vector. The expression vector is introduced into a host to produce the antibody (see European Patent Application Publication No. EP239400 and International Publication No. WO92-19759).

[0069] The FRs of a human antibody linked to the CDRs are selected so that the complementarity-determining regions can form a functional antigen-binding site. One or more amino acids in the framework regions of the antibody variable region may be optionally substituted, if necessary, so that the complementarity-determining regions of the reshaped human antibody can form a functional antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0070] Chimeric and humanized antibodies have the C region of a human antibody. The C region of a human antibody includes Cγ. For example, Cγ1, Cγ2, Cγ3, or Cγ4 can be used. The C region of a human antibody can also be modified to improve antibody stability or antibody production.

[0071] A chimeric antibody consists of the variable region of an antibody derived from a non-human mammal and the C region of a human antibody. A humanized antibody consists of the complementarity-determining region of an antibody derived from a non-human mammal and the framework and C regions of a human antibody. Both chimeric and humanized antibodies have low antigenicity in the human body and are therefore useful antibodies for use in the present invention.

[0072] Known methods for providing human antibodies include, in addition to previously described methods, techniques for obtaining human antibodies by panning human antibody libraries. For example, the variable regions of human antibodies can be expressed on the surface of phages in the form of single-chain antibodies (scFvs) by phage display, and phages that bind to the antigen of interest can be selected. The selected phages are genetically analyzed by sequencing the DNA encoding the variable regions of the human antibodies that bind to the antigen of interest. Once the DNA sequence of the scFv that binds to the antigen is determined, an expression vector suitable for the sequence can be created. The expression vector can be used to obtain human antibodies. These methods are well known and are described in, and can be found in, WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388.

[0073] The antibody gene constructed as described above can be expressed using any known method to obtain an antibody. For mammalian cells, the antibody can be expressed using DNA obtained by operably linking a commonly used and useful promoter, the antibody gene to be expressed, and a poly(A) signal downstream of the 3' end of the antibody gene, or a vector containing the DNA. Examples of promoters / enhancers include the human cytomegalovirus immediate-early promoter / enhancer.

[0074] Other promoters / enhancers that can be used for antibody expression in the present invention include viral promoters / enhancers such as those from retroviruses, polyoma viruses, adenoviruses, and simian virus 40 (SV40), as well as promoters / enhancers derived from mammalian cells, such as human elongation factor 1 alpha (HEF1α).

[0075] For example, the SV40 promoter / enhancer can be easily used according to the method of Mulligan et al. (Mulligan, R.C. et al., Nature (1979) 277, pp. 108-114), and the HEF1α promoter / enhancer can be easily used according to the method of Mizushima et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, pp. 532).

[0076] For E. coli, antibodies can be expressed by operably linking a commonly used and useful promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed. Examples of promoters include the lacZ promoter and the araB promoter. The lacZ promoter may be used according to the method of Ward et al. (Ward, E.S. et al., Nature (1989) 341, pp. 544-546; Ward, E.S. et al., FASEB J. (1992) 6, pp. 2422-2427), and the araB promoter may be used according to the method of Better et al. (Better, M. et al., Science (1988) 240, pp. 1041-1043).

[0077] The antibody secretion signal sequence used for production in the periplasm of E. coli may be the 1B signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379-4383). Antibodies produced in the periplasm are separated, followed by proper refolding of the antibody structure (see, for example, WO96 / 30394).

[0078] Replication origins that can be used include those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. Furthermore, the expression vector may contain a selection marker such as an aminoglycoside phosphotransferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or a dihydrofolate reductase (dhfr) gene to amplify the number of gene copies in the host cell system.

[0079] Any production system can be used to produce the antibodies used in the present invention. Production systems for antibody production include in vitro production systems and in vivo production systems. In vitro production systems include production systems using nuclear or prokaryotic cells.

[0080] Production systems using eukaryotic cells include those using animal, plant, or fungal cells. Animal cells known to be used include (1) mammalian cells, such as CHO, COS, myeloma, baby hamster kidney (BHK), Hela, and Vero; (2) amphibian cells, such as Xenopus oocytes; and (3) insect cells, such as sf9, sf21, and Tn5. Plant cells known to be used include cells derived from Nicotiana tabacum, which can be used for callus culture. Fungal cells known to be used include yeasts such as Saccharomyces, including Saccharomyces cerevisiae, and filamentous bacteria such as Aspergillus, including Aspergillus niger. Production systems using prokaryotic cells include those using bacterial cells. Bacterial cells known to be used include E. coli and Bacillus subtilis.

[0081] These cells are transformed with the desired antibody gene, and the transformed cells are cultured in vitro to obtain antibodies. Culturing is performed according to any known method. For example, media that can be used include DMEM, MEM, RPMI1640, and IMDM. Serum complements such as fetal calf serum (FCS) can also be used. Cells transfected with the antibody gene can also be injected into the peritoneal cavity of an animal to produce antibodies in vivo. On the other hand, in vivo production systems include production systems using animals and plants, and production systems using animals include production systems using mammals or insects.

[0082] Mammals that can be used include goats, pigs, sheep, mice, and cattle (Vicki Glaserm, SPECTRUM Biotechnology Applications, 1993). Insects that can be used include silkworms. Plants that can be used include, for example, tobacco.

[0083] Antibodies are produced and recovered from animals or plants into which antibody genes have been introduced. For example, the antibody gene is interrupted by a gene encoding a protein specifically produced in milk, such as goat beta-casein, to prepare a fusion gene. A DNA fragment containing the fusion gene carrying the antibody gene is transferred into a goat embryo. The embryo is then implanted into a female goat. The desired antibody is obtained from the milk produced by the goat that received the embryo (transgenic goat) or its offspring. Any appropriate hormone can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, pp. 699-702).

[0084] Silkworms can also be used to obtain desired antibodies by infecting them with a baculovirus containing the inserted antibody gene of interest and then collecting the infected silkworm's hemolymph (Maeda, S. et al., Nature (1985) 315, 592-594). Furthermore, when tobacco is used, the antibody gene of interest is inserted into a plant expression vector such as pMON530, and the vector is then introduced into bacteria such as Agrobacterium tumefaciens. The bacteria are then used to infect tobacco plants such as Nicotiana tabacum, and the desired antibody is obtained from tobacco leaves (Julian, KC Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

[0085] When antibodies are produced in the in vitro or in vivo production systems described above, host cells may be co-transformed with expression vectors into which DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) have been separately inserted, or may be transformed with a single expression vector into which DNAs encoding the H chain and L chain have been inserted (see International Publication No. WO94-11523).

[0086] The antibodies produced and expressed as described above may be separated from intracellular or extracellular components or the host and purified to homogeneity. The antibodies used in the present invention may be separated and purified by affinity chromatography. Columns used in affinity chromatography include, for example, Protein A columns and Protein G columns. Supports for Protein A columns include, for example, Hyper D, POROS, and Sepharose FF. Other details are not particularly limited, as long as they are used in methods for separating and purifying conventional proteins.

[0087] For example, the antibodies used in the present invention may be separated and purified by appropriately selecting or combining chromatography other than the above-described affinity chromatography, such as filtering, ultrafiltration, salting out, or dialysis. Chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, and gel filtration. These chromatographies are applicable to high-performance liquid chromatography (HPLC). Reverse-phase HPLC may also be used.

[0088] The concentration of the antibody obtained above can be measured by measuring absorbance, ELISA, etc. More specifically, in measuring absorbance, the antibody is appropriately diluted with PBS(-), and then the absorbance at 280 nm is measured. The absorbance at a concentration of 1 mg / ml is set to 1.350 D to calculate the antibody concentration. In ELISA, the antibody concentration can be measured as follows: 100 μl of goat anti-human IgG (manufactured by TAG) diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6) is added to a 96-well plate (manufactured by Nunc) and incubated overnight at 4°C to allow the antibody to be immunized. After blocking, 100 μl of the antibody used in the present invention, or a sample containing the antibody appropriately diluted, or human IgG (manufactured by CAPPEL) as a reference standard is added and incubated at room temperature for 1 hour.

[0089] After washing, 100 μl of 5000-fold diluted alkaline phosphatase-labeled human IgG (BioSource) was added and incubated at room temperature for 1 hour. After washing, substrate solution was added and incubated. The concentration of the target antibody was then calculated by determining the absorbance at 405 nm using a Microplate Reader Model 3550 (Bio-Rad).

[0090] The antibody used in the present invention may be an antibody conjugated with any of a variety of molecules, including polyethylene glycol (PEG), radioactive substances, and toxins. Such conjugated antibodies can be obtained by chemically modifying the antibodies produced as described above. Methods for modifying antibodies are established in the art. The term "antibody" in the present invention also includes conjugated antibodies.

[0091] Antibodies according to the present invention include not only bivalent antibodies such as IgG, but also monovalent antibodies and multivalent antibodies such as IgM. Multivalent antibodies according to the present invention include multivalent antibodies in which all antigen-binding sites are the same, and multivalent antibodies in which some or all of the antigen-binding sites are different.

[0092] The antibody according to the present invention may be a low molecular weight antibody. Low molecular weight antibodies include whole antibodies, such as antibody fragments lacking a part of whole IgG.

[0093] Antibody fragments can be produced by enzymatic digestion of antibodies. Enzymes known to produce antibody fragments include, for example, papain, pepsin, and plasmin. Alternatively, antibody fragments can be produced by constructing DNA encoding the antibody fragment, inserting the DNA into an expression vector, and then expressing the expression vector in a suitable host cell (see, for example, Co MS et al., J. Immunol. (1994) 152, pp. 2968-2976; Better M. & Horwitz AH, Methods in Enzymology (1989) 178, pp. 476-496; Pluckthun A. & Skerra A., Methods in Enzymology (1989) 178, pp. 497-515; Lamoyi E., Methods in Enzymology (1986) 121, pp. 652-663; Rousseaux J. et al., Methods in Enzymology (1986) 121, pp. 663-669; Bird RE & Walker BW, Trends Biotechnol. (1991) 9, pp. 132-137).

[0094] Each enzyme for digestion cleaves the antibody at a specific location, resulting in antibody fragments with specific structures as described below. On the other hand, genetic engineering techniques can be used to delete any portion of the antibody: In papain digestion:Fab; In pepsin digestion: F(ab')2 or F(ab'); and In plasmin digestion: Facb

[0095] An scFv can be obtained by linking the VH and VL of an antibody. In an scFv, the VH and VL are linked by a linker, preferably a peptide linker (Huston JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, pp. 5879-5883). The VH and VL in an scFv can be derived from any of the antibodies described herein. The peptide linker linking the V regions is not particularly limited. For example, any single-chain peptide consisting of about 3 to 25 residues can be used as the linker.

[0096] V regions can be linked, for example, by PCR as described above. For linking V regions by PCR, DNA encoding the complete or desired partial amino acid sequence encoded by the DNA sequence encoding the antibody H or H-chain V region and the DNA sequence encoding the antibody L or L-chain V region is used as a template. Each DNA encoding the H and L-chain V region is amplified by PCR using primers having sequences corresponding to the sequences at both ends of the DNA to be amplified. DNA encoding a peptide linker is then prepared. DNA encoding a peptide linker can also be synthesized by PCR. The primers used in this PCR have base sequences that can bind to each of the V region amplification products, which have been separately synthesized and previously added to the 5' end of the primer. Next, a PCR reaction is performed using each of the DNAs [VH DNA], [peptide linker DNA], and [VL DNA] together with primers for assembly PCR. The primers for assembly PCR are a combination of a primer that can anneal to the 5' end of [VH DNA] and a primer that can anneal to the 3' end of [VL DNA]. In other words, the assembly PCR primers comprise a set of primers that can be used to amplify DNA encoding the complete sequence of the scFv to be synthesized. The peptide linker DNA has a pre-added base sequence that can bind to each of the V region DNAs. The assembly PCR primers are used to link these DNAs and ultimately produce a full-length scFv as an amplification product. Once the DNA encoding the ScFv has been produced, an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained using any conventional method. The scFv can also be obtained by culturing the resulting recombinant cells and allowing the cells to express the DNA encoding the ScFv.

[0097] A diabody is a bivalent, low-molecular-weight antibody constructed by gene fusion (Holliger P. et al., Proc. Natl. Acad. Sci. USA (1993) 90, pp. 6444-6448, EP404097, WO93 / 11161). Diabodies are dimers consisting of two polypeptide chains. Generally, each polypeptide chain comprising a dimer is connected into a single chain by a linker between the VL and VH. In diabodies, the linker between the polypeptide chains is typically too short to connect the VL and VH on the same chain. Specifically, the amino acid residues comprising the linker preferably contain 2 to 12 residues, more preferably 3 to 10 residues, and particularly about 5 residues. Therefore, VL and VH encoded by a single polypeptide chain cannot form scFvs; therefore, two separate polypeptide chains dimerize to form two Fvs. As a result, diabodies have two antigen-binding sites.

[0098] sc(Fv)2 is a single-chain low-molecular-weight antibody in which two VHs and two VLs are linked by a linker (Hudson PJ & Kortt AA, J. Immunol. Methods (1999) 231, pp. 177-189). For example, sc(Fv)2 can be produced by linking two scFvs with a linker. Alternatively, sc(Fv)2 can be produced by linking two VHs and two VLs, starting from the N-terminus of a single-chain polypeptide, with a linker in the following order: [VH]-[linker]-[VL]-[linker]-[VH]-[linker]-[VL]. It should be noted that the order of the two VHs and two VLs is not particularly limited to the order described above, and any order is acceptable. For example, the order described below may also be included. [VL]-[linker]-[VH]-[linker]-[VH]-[linker]-[VL] [VH]-[linker]-[VL]-[linker]-[VL]-[linker]-[VH] [VH]-[linker]-[VH]-[linker]-[VL]-[linker]-[VL] [VL]-[linker]-[VL]-[linker]-[VH]-[linker]-[VH] [VL]-[linker]-[VH]-[linker]-[VL]-[linker]-[VH] The multiple linkers may be of the same type or of different types.

[0099] Linkers that can be used to link antibody variable regions include any peptide linker that can be incorporated by genetic engineering or a synthetic compound linker (e.g., the linkers disclosed in Protein Engineering (1996) 9, pp. 299-305). Peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art for any purpose. Generally, the amino acid residues comprising the peptide linker contain 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (e.g., 15 amino acids). The amino acid sequence comprising the peptide linker may be any sequence as long as it does not inhibit the binding activity of scFv.

[0100] Alternatively, a synthetic compound linker (chemical cross-linking agent) can be used to link the V regions. Cross-linking agents commonly used for cross-linking peptide compounds can be used in the present invention. Cross-linking agents that can be used include, for example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0101] <Monoclonal antibody that inhibits microtubule-dynamin 1 binding> The present invention also encompasses monoclonal antibodies that inhibit the binding of microtubules to dynamin 1 protein. Monoclonal antibodies against dynamin 1 protein are useful as monoclonal antibodies of the present invention. Non-limiting examples include monoclonal antibodies against proteins corresponding to dynamin 1 protein (human: SEQ ID NO: 1, mouse: SEQ ID NO: 2), i.e., monoclonal antibodies against a portion or the entire dynamin 1 protein. The sequence identity may or may not be completely identical to a portion or the entire dynamin 1 protein; for example, a sequence identity of at least 80%, preferably at least, preferably at least 8.5%, more preferably at least 90%, and even more preferably at least 95% is useful. Among these, monoclonal antibodies against the pleckstrin homology domain of dynamin 1 (human: SEQ ID NO: 3, mouse: SEQ ID NO: 4), the proline-rich domain of dynamin 1 (human: SEQ ID NO: 5, mouse: SEQ ID NO: 6), and peptides containing all or part of the amino acid sequences of SEQ ID NOs: 7, 8, and 9 are preferred. Monoclonal antibodies against peptides containing the amino acid sequences of SEQ ID NOs: 7, 8, and 9 are even more preferred.

[0102] For methods for producing monoclonal antibodies and other detailed descriptions, the descriptions in the section "Therapeutic agents for Alzheimer's disease containing inhibitors of dynamin 1-microtubule binding" can be applied as is.

[0103] Methods for Treating Alzheimer's Disease The method for treating Alzheimer's disease of the present invention is characterized by administering to a patient a peptide that inhibits the binding of dynamin-1 protein to microtubules. According to the method for treating Alzheimer's disease, the binding of dynamin-1 to microtubules is inhibited, thereby preventing the disruption of signal transduction by tau protein by administering to the patient a peptide that inhibits the binding of dynamin-1 protein to microtubules. As a result, endocytosis and signal transduction can be maintained almost normally, and Alzheimer's disease can be improved and treated. The method for treating Alzheimer's disease of the present invention may also be described as a method for administering to a patient a therapeutic agent for Alzheimer's disease of the present invention.

[0104] The peptide that inhibits the binding of dynamin 1 protein to microtubules is not particularly limited as long as it inhibits the binding of dynamin 1 protein to microtubules. Examples include peptides whose amino acid sequence partially matches that of dynamin 1. Among these, peptides containing a partial amino acid sequence of the pleckstrin homology domain of dynamin 1 and a partial amino acid sequence of the proline-rich domain of dynamin 1 are preferred. Peptides containing the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 are more preferred, and peptides containing the amino acid sequence of SEQ ID NO: 7 are even more preferred. Furthermore, it is also preferable to add a polypeptide, amino acid motif sequence, or the like to the above peptides to increase membrane permeability and promote brain translocation. Specific examples of polypeptides and amino acid motif sequences for increasing membrane permeability and promoting brain translocation include peptides containing the sequences represented by SEQ ID NOs: 10 to 15. Furthermore, peptides represented by SEQ ID NOs: 16 to 27 are specific examples of peptides to which a polypeptide, amino acid motif sequence, or the like for increasing membrane permeability and promoting brain translocation is added.

[0105] The same explanation as for therapeutic agents for Alzheimer's disease can be applied to peptides that inhibit the binding of dynamin-1 protein to microtubules.

[0106] A suitable daily dose of the above-mentioned peptide in the method for treating Alzheimer's disease of the present invention may be in the range of 0.005 mg to 500 mg per kg of body weight per day (e.g., 0.005 mg / kg to 100 mg / kg, 0.005 mg / kg to 30 mg / kg, 0.005 mg / kg to 1 mg / kg, 0.01 mg / kg to 30 mg / kg, 0.01 mg / kg to 3 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.02 mg / kg to 5 mg / kg, 0.02 mg / kg to 2 mg / kg, or 0.02 mg / kg to 1 mg / kg).

[0107] <Method for screening substances effective in treating Alzheimer's disease> A method for screening for a substance effective in treating Alzheimer's disease is characterized by comprising a step of measuring the inhibitory activity of a test substance on the binding of dynamin-1 protein to microtubules.

[0108] Specifically, the screening method is characterized by measuring the amount of binding between dynamin 1 protein and microtubules (tubulin) in the presence and absence of a test substance, and determining the presence or absence of the binding inhibitory activity of the test substance from the ratio of the amount of binding. Such a screening method of the present invention preferably includes the following steps 1) to 3).

[0109] 1) In the binding test of dynamin-1 protein and microtubules (tubulin), the amount of binding of both is measured when a test substance is added. 2) A process that measures the amount of binding between the two in the absence of a test substance. 3) The process of comparing the measurements from step 1) above with the measurements from step 2) above.

[0110] In step 1) of the screening method of the present invention, the test substance is first brought into contact with dynamin-1 protein and / or microtubules (tubulin) by adding the test substance to a binding test.

[0111] The dynamin 1 protein used in the screening methods of the present invention can be prepared using Escherichia coli, insect cells, wheat germ cell-free expression systems, etc. Furthermore, the species of dynamin 1 protein can be selected according to the target of the therapeutic agent for Alzheimer's disease, but mammals and humans are preferred. Furthermore, microtubules can be prepared by polymerizing tubulin purified from the brains of mammals such as pigs. The dynamin 1 protein and microtubules (tubulin) used in the screening methods of the present invention include, but are not limited to, mutants, alleles, variants, homologs, partial peptides, fusion proteins with other proteins, and those labeled with tags.

[0112] The species from which the dynamin-1 protein and microtubules (tubulin) used in the screening method of the present invention are derived is not limited to a particular species, and examples include humans, monkeys, mice, rats, guinea pigs, pigs, cows, yeast, and insects.

[0113] The state of the dynamin-1 protein and microtubules (tubulin) used in the screening method of the present invention is not particularly limited, and may be, for example, purified, expressed in cells, expressed in cell extracts, etc.

[0114] The test substance in the present invention is not particularly limited, and examples of the substance include single substances such as natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins (including antibodies), peptides, etc.; expression products of compounds, nucleic acids, peptides, and gene libraries; cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single-cell extracts, and animal cell extracts. The test substance in the present invention may also be a mixture of these substances. These test substances can also be labeled, if necessary, before use. Examples of labels include radioactive labels and fluorescent labels.

[0115] In the present invention, contact is performed according to the state of dynamin-1 protein and microtubules (tubulin). For example, when dynamin-1 protein and microtubules (tubulin) are purified, this can be performed by adding the test substance to the purified preparation. Furthermore, when the test substance is expressed in cells or cell extracts, the test substance can be added to the cell culture medium or cell extract, respectively, or can be directly administered to the experimental animal. If the test substance is a protein, contact can also be performed by, for example, introducing a vector containing DNA encoding the protein into cells expressing dynamin-1 protein and microtubules (tubulin), or adding the vector to a cell extract expressing dynamin-1 protein and microtubules (tubulin).

[0116] In the present invention, a test substance may be contacted with dynamin-1 protein and microtubules (tubulin) by adding the test substance to a sample containing dynamin-1 protein and microtubules (tubulin). Alternatively, the test substance may be added to a sample containing either dynamin-1 protein or microtubules (tubulin), and then the other sample without the test substance may be added and contacted with the test substance.

[0117] In the present invention, the amount of binding between dynamin 1 protein and microtubules (tubulin) is then measured. In step 2) of the screening method of the present invention, the amount of binding between dynamin 1 protein and microtubules (tubulin) is measured when a test substance is not added to a sample containing dynamin 1 protein and microtubules (tubulin). Specifically, the amounts of binding between dynamin 1 protein and microtubules (tubulin) are measured when a test substance is added (contacted) and when a test substance is not added (contacted), and the values ​​are compared in step 3) of the screening method. If the amount of binding of dynamin 1 protein to microtubules (tubulin) is reduced when the test substance is added compared to when the test substance is not added, the test substance can be determined to be effective in inhibiting the binding of dynamin 1 protein and microtubules (tubulin).

[0118] For example, a microtubule co-sedimentation assay can be used to measure the amount of binding between dynamin 1 protein and microtubules (tubulin). In the microtubule co-sedimentation assay, dynamin 1 protein bound to microtubules is pelleted together with the microtubules during centrifugation. The presence of a test substance that inhibits the binding of dynamin 1 protein to microtubules reduces the amount of dynamin 1 protein that pellets together, and the degree of this phenomenon can be used to determine the binding inhibitory effect. [Example]

[0119] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0120] Materials and Methods animal All experiments were conducted in accordance with the guidelines of the Physiological Society of Japan and the animal experimentation regulations of the Okinawa Institute of Science and Technology Graduate University.

[0121] Recombinant human tau preparation Human tau (h-tau) lacking the MT-binding domain (amino acids 244–367, del-MTBD) was generated by site-directed mutagenesis as previously described (40). Wild-type (WT) and del-MTBD mutant h-tau of the 0N4R isoform were expressed in Escherichia coli (BL21 / DE3) and purified as previously described (73) with minor modifications. Briefly, harvested bacteria expressing recombinant tau were lysed in homogenization buffer (50 mM PIPES, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 5 μg / ml leupeptin, pH 6.4), sonicated, and centrifuged at 27,000 × g for 15 min. The supernatant was applied to a phosphocellulose column (P11, Whatman). After washing with homogenization buffer containing 0.1 M NaCl, the h-tau-containing fraction was eluted with buffer containing 0.3 M NaCl. The protein was then precipitated with 50% saturated ammonium sulfate and resolubilized in homogenization buffer containing 0.5 M NaCl and 1% 2-mercaptoethanol. After incubation at 100°C for 5 min, the heat-stable (soluble) fraction was obtained by centrifugation at 21,900 × g and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) using Cosmosil Protein-R (Nacalai tesque Inc.). Aliquots of the h-tau-containing fraction were lyophilized and stored at -80°C. The purified h-tau protein was quantified by SDS-PAGE followed by Coomassie Brilliant Blue staining.

[0122] Purification of recombinant human dynamin 1 protein His-tagged human dynamin 1 was expressed using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific, Waltham, MA, USA) and purified as previously described ( 74 ). The purified dynamin solution was concentrated using Centriplus YM50 (cat#4310; Merck-Millipore, Darmstadt, Germany).

[0123] Microtubule polymerization assay The effects of tau and nocodazole on MT polymerization were tested using a tubulin polymerization assay (Cytoskeleton Inc., Denver, CO). Briefly, purified wild-type or del-MTBD mutant h-tau (10 μM) was mixed with porcine tubulin (20 μM) in assembly buffer at 37°C. Nocodazole was added to the mixture at 0 min of incubation. MT polymerization was assayed fluorometrically (excitation 360 nm, emission 465 nm) at 1-min intervals for 30 min using an Infinit F-200 Microplate Reader (TECAN, Mannedorf, Switzerland). After incubation, the resulting solution was centrifuged at 100,000 × g for 15 min at 20°C. The supernatant (free tubulin fraction) and pellet (microtubule fraction) were subjected to SDS-PAGE to quantify the amount of tubulin assembled into MTs.

[0124] Peptide synthesis and LC-MS / MS analysis Peptides were synthesized by conventional 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) using a ResPep SL automated peptide synthesizer (Intavis Bioanalytical Instruments) with preloaded Fmoc-alanine TCP resin (Intavis Bioanalytical Instruments). All Fmoc amino acids were purchased from Watanabe Chemical Industries and prepared at 0.5 M in N-methylpyrrolidone (NMP, Wako Pure Chemical Industries). After synthesis, peptides were cleaved with (v / v / v) 92.5% TFA, 5% TIPS, and 2.5% water for 2 h, precipitated with t-butyl methyl ether at -30 °C, pelleted, resuspended in water, and lyophilized overnight (EYELA FDS-1000). The purity and sequence of all synthesized peptides were then confirmed by LC-MS / MS using an Ultimate 3000 nano-HPLC system (Dionex), an HTC-PAL autosampler (CTC Analytics), and a Q-Exactive Plus Orbitrap hybrid mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source.

[0125] MT-dynamin binding assay Microtubule-binding assays were performed using a Microtubule-Associated Protein Spin-Down Assay Kit (cat# BK029, Cytoskeletion Inc., Denver, CO, USA). Briefly, 20 μl of 5 mg / ml tubulin in general tubulin buffer (GTB; 80 mM PIPES pH 7.0, 2 mM MgCl, 0.5 mM EGTA) supplemented with 1 mM GTP was added to cushion buffer (80 mM PIPES pH 7.0, 1 mM MgCl). l2Polymerization was initiated by adding 2 μl of 1 mM EGTA, 60% glycerol (1 mM EGTA, 60% glycerol) and incubation at 35°C for 20 min. Microtubules were stabilized with 20 μM taxol. Taxol-stabilized microtubules (2.5 μM) and dynamin 1 (1 μM) were incubated with or without 1 mM peptide in GTB for 30 min at room temperature. After incubation, 50 μl of the mixture was added on top of 100 μl of cushion buffer supplemented with 20 μM taxol and then centrifuged at 100,000 × g for 40 min at room temperature. After ultracentrifugation, 50 μl of the supernatant was collected and mixed with 10 μl of 5x sample buffer. The resulting pellet was resuspended in 50 μl of 1x sample buffer. 20 μl of each sample was analyzed by SDS-PAGE and stained with SYPRO Orange. Protein bands were visualized using an FLA-3000 (FUJIFILM Co., Ltd., Tokyo, Japan).

[0126] Immunohistochemical analysis The following primary antibodies were used: anti-β3-tubulin (Synaptic System, #302304), anti-human tau (BioLegend, #806501), and anti-dynamin (Invitrogen, PA1-660). Secondary antibodies were goat IgG conjugated with Alexa Fluor 488, 568, or 647 (Thermo Fisher Scientific). Acute brainstem sections (175 μm thick, see below) were fixed with 4% paraformaldehyde in PBS for 30 min at 37°C and overnight at 4°C. The next day, sections were rinsed three times with PBS, permeabilized with 0.5% Triton X-100 (Tx-100; Nacalai Tesque) in PBS for 30 min, and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) and 0.05% Tx-100 in PBS for 45 min. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS 0.05% Tx-100, 0.3% BSA. The next day, sections were rinsed three times for 10 minutes with PBS containing 0.05% Tx-100 and incubated for 1 hour at room temperature (RT) with the corresponding secondary antibodies diluted in PBS 0.05% Tx-100, 0.3% BSA. Sections were rinsed three more times for 10 minutes with PBS 0.05% Tx-100 and finally washed for another 10 minutes with PBS. Finally, sections were mounted on glass slides (Matsunami) using liquid mounting medium (Ibidi) and sealed with nail polish. Confocal images were acquired with a laser scanning microscope (LSM780 or LSM900, Carl Zeiss) equipped with a Plan-Apochromat 63x oil immersion objective (1.4 NA) and 488, 561, and 633 nm excitation laser lines. To quantify the fluorescence intensity levels, regions of interest were delimited around the calyx termini and background fluorescence was subtracted using ImageJ software.

[0127] GST protein purification A cDNA (NM_004408.4) (75) encoding the PH domain (amino acids 521–618) of human dynamin 1 was prepared by PCR and subcloned into the pGEX-6P vector. The resulting plasmid was transformed into the bacterial strain BL21(DE3)pLysS for protein expression. Expression of the GST fusion protein was amplified by 0.1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) in LB medium supplemented with 100 μg / ml ampicillin at 37°C for 3–6 h. 600 The GST-fusion proteins were induced at a ribosomal ratio of 0.8. The GST-fusion proteins were then purified as described (76). The nucleotide sequences of the constructs used in this study were verified by DNA sequence analysis. All purified protein solutions (1–3 mg / ml) were stored at −80°C and thawed at 37°C before use.

[0128] Microscopic observation of microtubules and GST-PH protein GST or GST-PH was labeled using a HiLyte Fluor-555 labeling kit (cat#LK14, Dojindo Co. LTD, Kumamoto, Japan) according to the manufacturer's instructions. HiLyte Fluor-555-labeled GST or GST-PH was mixed with unlabeled proteins at a 1:1.2 ratio. Flutax1-stabilized microtubules (4.1 μM) and 11 μM fluorescent GST or GST-PH were mixed in GTB containing 2 μM Flutax1 at 37°C for 60 minutes. 8 μl of the mixture was dropped onto a glass slide and mounted with Fluoromount (cat#K024, Diagnostic BioSystems, CA, USA). Samples were examined using a spinning-disk confocal microscope system (X-Light Confocal Imager; CREST OPTICS SPA, Rome, Italy) combined with an inverted microscope (IX-71; Olympus Optical Co., Ltd., Tokyo, Japan) and an iXon+ camera (Oxford Instruments, Oxfordshire, UK). The confocal system was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). When necessary, images were processed using Adobe Photoshop CS3 or Illustrator CS3 software. For electron microscopy, samples were negatively stained for imaging with a 120 kV transmission electron microscope (TEM) (H-7650, Hitachi High-Tech Corp., Tokyo, Japan).

[0129] Slice electrophysiology Male and female C57BL / 6N mice (postnatal days 13-15) were killed by decapitation under isoflurane anesthesia. The brainstem was isolated and transverse sections (175 μm thick) containing the medial nucleus of the trapezoid body (MNTB) were cut into Ca sections. 2+ Reduce the concentration (0.1 mM), Mg 2+Sections were cut using a vibratome (VT1200S, Leica) in increasing concentrations (3 mM) of ice-cold artificial cerebrospinal fluid (aCSF, see below) or sucrose-based aCSF (NaCl was replaced with 300 mM sucrose, and CaCl2 and MgCl2 concentrations were 0.1 mM and 6 mM, respectively). Sections were incubated at 36–37°C for 1 h in standard aCSF (pH 7.4, 310–320 mOsm when bubbled with 95% O2 and 5% CO2) containing (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaHPO4, 2 CaCl2, 1 MgCl2, 10 glucose, 3 myo-inositol, 2 sodium pyruvate, and 0.5 sodium ascorbate.

[0130] Whole-cell recordings were made from presynaptic terminals and postsynaptic MNTB principal neurons visually identified with a 60x or 40x water-immersion objective (LUMPlanFL, Olympus) attached to an upright microscope (Axioskop2, Carl Zeiss, or BX51WI, Olympus, Japan) using a patch-clamp amplifier (Multiclamp 700A, Molecular Devices, USA for paired recordings, EPC-10 USB, HEKA Elektronik, Germany for presynaptic capacitance measurements). Data were acquired at a sampling rate of 50 kHz using pClamp (for Multiclamp 700A) or Patchmaster software (for EPC-10 USB) after online filtering at 5 kHz. Presynaptic pipettes were pulled against a resistance of 7–10 MΩ, and the series resistance was 14–20 MΩ (compensated 70% to achieve a final value of 7 MΩ). The resistance of the postsynaptic pipette was 5–7 MΩ, and its series resistance was 10–25 MΩ (compensated up to 75% for a final value of 7 MΩ). AThe solution contained picrotoxin (10 μM) and strychnine hydrochloride (0.5 μM) to block the ATP and glycine receptors, respectively. The postsynaptic pipette solution contained (in mM): 130 CsCl, 5 EGTA, 1 MgCl, 5 QX3 14-Cl, and 10 HEPES (adjusted to pH 7.3–7.4 with CsOH). The presynaptic pipette solution contained (in mM): 10 5 K methanesulfonic acid, 30 KCl, 40 HEPES, 0.5 EGTA, 1 MgCl, 12 phosphocreatine (Na salt), 3 ATP (Mg salt), and 0.3 GTP (Na salt) (adjusted to pH 7.3–7.4 with KOH, 315–320 mOsm).

[0131] For simultaneous presynaptic and postsynaptic whole-cell recordings, postsynaptic MNTB neurons were voltage-clamped at a holding potential of -70 mV, and EPSCs were elicited at 0.1 Hz or 1 Hz by action potentials triggered by a depolarizing current (1 ms) injected into the calyx terminal. For intraterminal delivery of taxol (1 μM), taxol was diluted into the presynaptic pipette solution from a 5 mM DMSO stock concentration to a final DMSO concentration of 0.02%. Similarly, nocodazole (20 μM, 0.1% DMSO) was included in the presynaptic pipette solution. The presynaptic pipette solution for the nocodazole control contained 0.1% DMSO. For simultaneous presynaptic and postsynaptic recordings, WT h-tau, del-MTBD tau, taxol, or synthetic peptides were delivered to the calyx terminal using the pipette perfusion method (42, 77). Briefly, a thin perfusion tube made of plastic and glass tubing was attached to the presynaptic patch pipette. After pipette solution containing proteins and / or peptides was reintroduced into the tube, the tube was inserted into the patch pipette, with its tip positioned 500–600 μm posterior to the tip of the presynaptic patch pipette. After recording baseline EPSCs, the tube solution was delivered into the presynaptic patch pipette under positive pressure (8–10 psi) using a pico pump.

[0132] Membrane capacitance (C m) measurements were performed at RT in a whole-cell configuration from the calyx presynaptic terminal of Herth (47, 49). The calyx terminal was voltage-clamped at a holding potential of -80 mV, and a sinusoidal voltage command (1 kHz, peak-to-peak amplitude 60 mV) was applied. Presynaptic voltage-dependent Ca 2+ Current (I Ca To isolate tau, aCSF contained 10 mM tetraethylammonium chloride, 0.5 mM 4-aminopyridine, 1 μM tetrodotoxin, 10 μM bicuculline methiodide, and 0.5 μM strychnine hydrochloride. The presynaptic pipette solution contained the following (in mM): 125 Cs methanesulfonic acid, 30 CsCl, 10 HEPES, 0.5 EGTA, 12 disodium creatine phosphate, 3 Mg ATP, 1 MgCl2, and 0.3 Na2GTP (adjusted to pH 7.3 with CsOH, 315–320 mOsm). Tau or synthetic peptides were dissolved in the pipette solution, and the pipette was refilled immediately after filling the tip with tau-free pipette solution. Care was taken to maintain a series resistance <16 MΩ to allow dialysis of the distal end with the pipette solution. Coat the tip of the recording pipette with dental wax to minimize stray capacitance (4–5 pF). Use a single rectangular pulse (-80 to 10 mV, 20 ms duration) to record presynaptic I. ca In these experiments, the exocytic capacitance change (ΔCm) corresponded to approximately five times more SVs (estimated from ΔCm divided by the Cm of a single SV) than the number of SVs in the immediately releasable pool due to presynaptic action potentials (estimated by dividing the size of the maximal evoked EPSC by the size of the miniature EPSC). Membrane capacitance changes within 450 ms of square pulse stimulation were excluded from the analysis to avoid contamination by conductance-dependent capacitance artifacts (49). To avoid the effect of capacitance drift in the baseline, we used a baseline drift of 5 fFs measured 0–10 s before stimulation. -1 Data was excluded if the drift exceeded 1 to 5 fFs. -1 If <0.01, we subtracted the linear regression line of the baseline from the data for baseline correction. Endocytosis rates were calculated as the normalized C for the first 10 seconds after stimulation. m Calculated from the slope of change.

[0133] Data Analysis and Statistics Data were analyzed using IGOR Pro 6 (WebMatrics), Excel 2016 (Microsoft), and StatPlus (AnalystSoft Inc.), and KaleidaGraph for Macintosh, version 4.1 (Synergy Software Inc., Essex Junction, VT, USA). All values ​​are presented as mean ± SEM. Differences in paired or unpaired t-tests, one-way ANOVA with Scheffe post-hoc tests, and repeated measures two-way ANOVA with Scheffe post-hoc tests were considered statistically significant at p < 0.05.

[0134] <Result> Intraterminal delivery of WT h-tau impairs excitatory synaptic transmission To address whether elevated soluble h-tau levels in presynaptic terminals could affect synaptic transmission, we purified wild-type recombinant h-tau (0N4R) and its deletion mutant (del-MTBD) lacking the MT-binding site (244Gln to 367Gly) using an Escherichia coli expression system (Figure 1—Supplementary Figure 1A) (40). These recombinant h-tau proteins are highly soluble at room temperature and show no signs of granulation (41). In simultaneous presynaptic and postsynaptic recordings at the calyx of Held in mouse brainstem slices, we recorded EPSCs evoked at 1 Hz by presynaptic action potentials (Figure 1). After confirming stable EPSC amplitude for 10 min, we injected a large volume of internal solution containing WT h-tau (20 μM) into the presynaptic whole-cell pipette via the attached thin tubing, replacing most of the pipette solution and allowing h-tau to diffuse into the presynaptic terminal (illustrated in Figure 1A) (42, 43). After introducing h-tau (20 μM), the amplitude of glutamatergic EPSCs gradually decreased, reaching 23 ± 9% at 30 min (Figure 1A, p < 0.01, paired t-test, n = 6 synapses in 6 slices). WT h-tau introduced at a lower concentration (10 μM) resulted in a slower EPSC attenuation, reaching 65 ± 5% at 30 min (p < 0.01, n = 5 in 5 slices). Del-MTBD (20 μM), which lacks tubulin polymerization ability, introduced similarly (Figure 1—Supplementary Figure 1B), did not affect EPSC amplitude (Figure 1A). Because the h-tau concentration in the presynaptic terminal equilibrates with the concentration in the presynaptic whole-cell pipette, which has a much larger volume than the terminal ( 44 ), these results suggest that WT h-tau >10 μM can significantly impair excitatory synaptic transmission.

[0135] The inhibitory effect of WT h-tau on EPSCs was clearly frequency-dependent. When elicited at 0.1 Hz, WT h-tau (20 μM) produced a negligible decrease in EPSC amplitude (85 ± 12% at 30 min post-infusion, p = 0.21, n = 5; Figure 1B). Because taxol shares a common binding site with tau and MTs (45) and assembles tubulin into MTs (Figure 1—Supplementary Figure 1B), we tested the effect of taxol (1 μM) on EPSCs (Figure 1C). Similar to h-tau, taxol significantly attenuated EPSCs elicited at 1 Hz (41 ± 12 at 30 min, n = 5, p < 0.05), but not EPSCs elicited at 0.1 Hz (104 ± 3.0% at 30 min, n = 5, p = 0.60). Taken together, these results suggest that newly assembled MTs at presynaptic terminals by WT h-tau or taxol result in activity-dependent attenuation of excitatory synaptic transmission.

[0136] WT h-tau primarily inhibits SV endocytosis and secondarily inhibits exocytosis To determine the primary target of h-tau that causes synaptic dysfunction, we performed membrane capacitance measurements at the calyx of Held (46-49). Because stray capacitance in the perfusion pipette interferes with capacitance measurements, we prefilled only the tip of a conventional patch pipette with normal internal solution, then backfilled the patch pipette with h-tau to ensure GΩ seal formation. This resulted in a significant and variable delay in intra-terminal diffusion, and therefore no obvious effect was observed beyond 10 min after the whole-cell patch membrane was disrupted. Twenty min after whole-cell patch filling with WT h-tau (20 μM), endocytic capacitance showed a significant slowing (Figure 2), whereas the extent of exocytic capacitance induced by the depolarizing pulse (ΔC m ) or Ca 2+ Current charge (Q Ca ) was not different from the control without h-tau loading. After 30 min of h-tau loading, the endocytic capacitance change was further slowed (p<0.01), and the exocytic ΔC mshowed a significant decrease (p<0.05, n=5), and Q Ca These results suggest that the primary target of h-tau toxicity is synaptic vesicle (SV) endocytosis. Inhibition of endocytosis inhibits recycling SV replenishment, thereby reducing exocytic release of neurotransmitters as a secondary effect.

[0137] Inhibition of SV endocytosis and synaptic transmission by WT h-tau requires de novo MT assembly Because de novo MT assembly can occur after h-tau loading (Figure 1, Figure 1-Supplementary Figure 1), we tested whether the tubulin assembly inhibitor nocodazole could reverse the toxic effects of h-tau on SV endocytosis and synaptic transmission. In tubulin assembly assays, nocodazole inhibited h-tau-dependent MT assembly in a concentration-dependent manner, reaching maximum inhibition at 20 μM (Figure 3A). In presynaptic capacitance measurements, nocodazole (20 μM) coloaded with h-tau (20 μM) completely prevented h-tau toxicity on endocytosis (Figure 3B) and synaptic transmission (Figure 3C). Nocodazole alone (20 μM) had no effect on exo-endocytosis (Figure 3B) or EPSC amplitude (Figure 3C). It is likely that WT h-tau loaded into the calyx terminals assembled MTs de novo, thereby impairing SV endocytosis and synaptic transmission.

[0138] WT h-tau assembles MTs at the calyx ends and captures dynamin The monomeric GTPases dynamin 1 and 3 play important roles in the fission process of SV endocytosis (50-52). Because dynamin was originally discovered as a MT-binding protein (39), we hypothesized that newly formed MTs might capture free dynamin in the cytoplasm. If this were true, this would lead to an increase in MT-bound dynamin. To test this hypothesis, we performed immunofluorescence microscopy and image analysis to quantify MTs and dynamin. After whole-cell injection of h-tau into the calyx terminals, sections were chemically fixed and permeabilized to wash out cytoplasmic free molecules, such as tubulin monomers, from the terminals, thereby enhancing the signal from larger structures such as MTs or MT-bound molecules. A fluorescent h-tau antibody identified calyx terminals transfected with WT h-tau (20 μM, Figure 4A). Double staining with mouse β3 tubulin antibody revealed a 2.1-fold increase in MT signal at h-tau-introduced terminals compared to those without h-tau (p = 0.01, n = 5, unpaired two-tailed t-test with Welch's correction, Figure 4B). Triple labeling with dynamin antibody further revealed a 2.6-fold increase in dynamin signal (p = 0.01, n = 5, two-tailed t-test with Welch's correction, Figure 4B). Super-resolution imaging showed dynamin clustered with MTs at tau-introduced calyx terminals (Figure 4 - Supplementary Figure 1). These results suggest that soluble WT h-tau can assemble MTs at presynaptic terminals, thereby capturing cytoplasmic dynamin, which is essential for SV endocytosis.

[0139] In addition to dynamin, MTs can bind to various other proteins. Among them, the formin mDia can bind to MTs (53) and, together with F-actin, intersectin, and endophilin, is involved in endocytic scaffolding. Acute depolymerization of F-actin (38, 46) or genetic disruption of intersectin (54) does not affect SV endocytosis in the calyx of the Held. However, the formin mDia inhibitor SMFH2 has been reported to inhibit endocytosis in the calyx terminals of rats before the onset of auditory function (postnatal days [P] 8–12) (55). We reexamined whether this drug could inhibit SV endocytosis in the calyx terminals in slices from mice after the onset of auditory function (P13–14). SMFH2 slightly prolonged SV endocytosis, but this effect was not statistically significant (Figure 4—Supplementary Figure 2A). Therefore, formins are unlikely to contribute significantly to the significant endocytic slowing observed after intraterminal tau introduction (Fig. 2 ).

[0140] Additionally, because endophilin is involved in clathrin uncoating, which is required for glutamate-induced SV re-entry (57), it is possible that endophilin binding to MTs (56) may cause EPSC attenuation. Impaired SV re-entry during recycling reduces the amplitude and frequency of miniature EPSCs (58). However, neither amplitude nor frequency was affected by intraterminal injection of tau (20 μM) (Figure 4—Supplementary Figure 2B). Therefore, endophilin-MT binding is unlikely to underlie the EPSC attenuation caused by intraterminal tau injection (Figure 1).

[0141] Microtubule-dynamin binding inhibitor peptide reduces h-tau toxicity on SV endocytosis and synaptic transmission To prevent the toxic effects of h-tau on endocytosis and signaling, we searched for dominant-negative (DN) peptides that block MT-dynamin binding. Because the MT-binding domain of dynamin is unknown, we synthesized 11 peptides from the pleckstrin homology (PH) domain of dynamin 1 and 11 peptides from the proline-rich domain (Figure 4-Supplementary Figure 1A) and subjected them to MT-dynamin 1 binding assays. Among the 22 peptides, one peptide corresponding to amino acids 560–571 of the PH domain (which we named "PHDP5") significantly inhibited MT-dynamin 1 interaction (Figure 5A, Figure 5-Supplementary Figure 1B and C). SYPRO Orange staining revealed dynamin 1 as a band of approximately 100 kDa in the precipitate (ppt), 1.7 ± 0.4%. In the presence of MT, dynamin 1 in the ppt increased to 22.6 ± 2.4%, indicating sequestration of dynamin 1 by MT. When PHDP5 was added to MT and dynamin 1, dynamin 1 in the ppt fraction decreased to 6.3 ± 2.4%, indicating that PHDP5 functions as a DN peptide that inhibits MT-dynamin interaction (Fig. 5A).

[0142] Cryo-electron microscopy of dynamin 1 assembled in lipid membranes revealed that the PH domain is tucked into the dynamin structure in the apo state, but upon GTP binding, it undergoes a conformational change that exposes it toward the membrane (59). Negative-stain electron micrographs show that dynamin 1 periodically aligns with the surface of MTs (60), suggesting a helical polymerization-like dynamin-membrane interaction (61). Therefore, the PH domain, including the putative binding site PHDP5, may be exposed on the surface of MTs. To examine whether the dynamin 1 PH domain can directly bind to MTs, immunofluorescently labeled MTs and the glutathione transferase PH domain (GST-PH) were mixed and observed by confocal and electron microscopy (Figure 5—Supplementary Figure 2). Confocal microscopy imaging revealed that GST-PH colocalized with MTs, in contrast to the control where MTs were mixed with GST alone (Figure 5—Supplementary Figure 2A). These results were further confirmed by electron microscopy imaging, which showed colocalization of MTs and GST-PH (Figure 5—Supplementary Figure 2B). Thus, although the dynamin 1 PH domain can bind to MTs, it is not yet clear whether PHDP5 can directly bind to MTs.

[0143] Introduction of PHDP5 (0.25 mM) alone into the calyx terminal did not affect exo-endocytic membrane capacitance changes, but when co-injected with WT h-tau (20 μM), it significantly attenuated h-tau-induced endocytic slowing (p<0.05, Figure 5B). A scrambled PHDP5 peptide (0.25 mM), introduced as a control, did not affect h-tau-induced endocytic slowing. Similar to its effect on capacitance changes, intra-terminal injection of PHDP5 alone (1 mM) did not affect EPSC amplitude, but when co-injected with WT h-tau (20 μM), it significantly attenuated the inhibitory effect of h-tau on EPSC amplitude (p<0.01, Figure 5C). Co-injection of scrambled PHDP5 (1 mM) with h-tau (20 μM) did not affect h-tau-induced EPSC attenuation (p=0.46). These results further support that WT h-tau leads to dynamin deficiency due to the de novo assembly of MTs, thereby impairing SV endocytosis and synaptic transmission. These results also highlight PHDP5 as a potential therapeutic tool to rescue synaptic dysfunction associated with AD or PD.

[0144] MTs were prepared from tubulin and stabilized with taxol. Taxol-stabilized MTs and Dyn1 were then incubated with or without PHDP5. Supernatant (unbound protein) and pellet (bound protein) fractions were separated using ultracentrifugation. Samples were analyzed by SDS-PAGE gel and visualized by SYPRO Orange staining. The results showed that the percentage of Dyn1 in the pellet fraction did not differ between the supernatant and pellet fractions after incubation with PHDP5. This may be due to competitive binding of Dyn1 / PHDP5 to MTs (Figure 6A).

[0145] We changed the incubation order to mix MT with PHDP5 and then incubate with Dyn1, and the results showed that the percentage of Dyn1 decreased in the pellet fraction in the presence of PHDP5 (Figure 6B).

[0146] To optimize the conditions for PHDP5 blocking MT-dynamin binding, we tested various PHDP5 concentrations and incubation times with Dyn1. The results showed that the percentage of Dyn1 in the pellet fraction was lower when MTs were incubated with 3 mM PHDP5 than when they were incubated with 1 mM PHDP5. The lower percentage of Dyn1 in the pellet fraction demonstrates its potential to inhibit MT-dynamin interaction (Figure 7A). When MTs were incubated with 1 mM PHDP5, a shorter incubation period with Dyn1 (10 min) resulted in a lower percentage of Dyn1 in the pellet fraction. Without PHDP5, there was no difference between the time points (Figure 7B). When MTs were incubated with 3 mM PHDP5, a 5-min incubation with Dyn1 resulted in a lower percentage of Dyn1 in the pellet fraction (Figure 7C). We prepared several FITC-labeled PHDP5 peptide candidates that improve the brain barrier permeability of PHDP5 (Table 3). The N-terminus of the construct was protected with β-alanine (βA) and labeled with FITC. Several FITC-labeled PHDP5 candidates and their crumbled controls were also tested under the same conditions. The first candidate (SEQ ID NO: 16) showed a lower % of Dyn1 than PHDP5 (Figure 7D). The results of statistical analysis are shown in Figures 8A and 8B.

[0147] [Table 3]

[0148] Electron images of negatively stained microtubules and dynamin 1 in the presence or absence of PHDP5 are shown in Figure 9. MT-dynamin 1 plus FP5#3 appears to stabilize the MT-dynamin 1 interaction.

[0149] <Consideration> Using the calyx of Held in brainstem slices as a mammalian AD model for analyzing central excitatory synaptic transmission, we demonstrated that intraterminal delivery of WT h-tau impairs vesicle endocytosis and synaptic transmission by de novo MT assembly. Previous overexpression studies in cultured cells reported MT assembly following injection or overexpression of WT tau (36, 62, 63) or phosphorylated tau (34, 64). Compared to overexpression, our whole-cell method allows targeted delivery of molecules to presynaptic terminals at defined concentrations due to the larger pipette-to-cell volume ratio (44). In postmortem brain tissue homogenates from AD patients, soluble tau content has been estimated at 6 ng per μg of protein, eightfold higher than in controls (65). Assuming the protein content in brain homogenates is 10%, the concentration of 60 kDa tau in AD patient brains is estimated to be 10 μM. Because elevated soluble tau concentrations may occur primarily in axons and axon terminal compartments of neurons, soluble tau concentrations in presynaptic terminals in AD patients may be higher. Our results in the calyx of Held suggest that excitatory synaptic transmission may generally be significantly impaired in such situations. Indeed, the degree of EPSC attenuation after administration of WT h-tau is comparable to that induced by clinical doses of the general anesthetic isoflurane in the calyx of Held in slices (48). In AD, tau pathology begins in the locus coeruleus of the brainstem and spreads transsynaptically to hippocampal and neocortical neurons (66). Our results in our model synapse suggest that synaptic function in such tau transmission pathways may be severely affected in the early stages of AD.

[0150] Membrane capacitance measurements in the calyx of Held revealed that SV endocytosis is the primary target of WT h-tau toxicity. Slowing endocytosis impairs SV recycling and reuse, thereby inhibiting SV exocytosis, particularly in response to high-frequency stimulation (49). The toxic effects of h-tau on SV endocytosis and synaptic transmission were prevented by coapplication of nocodazole. Combined with the lack of toxicity of del-MTBD and the toxic effects of Taxol on synaptic transmission, these results suggest a pathological role for overassembled MTs. Similar to WT h-tau, intraterminal introduction of WT α-synuclein slows SV endocytosis and impairs the fidelity of high-frequency neurotransmission in the calyx of Held (46). α-Synuclein toxicity can be rescued by inhibiting MT assembly with nocodazole or the photosensitive colchicine derivative PST-1. Thus, a common mechanism may underlie synaptic dysfunction in AD and PD. Compared to α-synuclein, h-tau is much more toxic to endocytosis and synaptic transmission, and therefore, abnormal elevation of endogenous molecules above homeostatic levels may lead to AD and PD symptoms, as in many other human diseases.

[0151] The GTPase dynamin, a well-known player in SV endocytic fission (50, 52), was originally discovered as a MT-binding protein (39). Subsequent studies showed that this interaction upregulates dynamin's GTPase activity (67, 68), can induce MT instability by dynamin 2 (69), or stabilizes MT bundling by dynamin 1 (60). However, the dynamin binding domain remained unidentified. In this study, calyx terminals transfected with WT h-tau exhibited a significant increase in immunofluorescence signal intensity corresponding to bound dynamin. This was associated with an increase in intraterminal MTs, suggesting that newly assembled MTs induced by h-tau transfection capture cytoplasmic dynamin. These results fully explain the impairment of SV endocytosis by intraterminal h-tau transfection. Through synthetic peptide screening, we found that a dodecapeptide derived from the dynamin 1 PH domain significantly inhibited MT-dynamin interaction. This peptide, PHDP5, is approximately 80% homologous to dynamin 3, another isoform involved in vesicle endocytosis (51). Although the direct binding of this peptide to MTs remains unclear, it significantly rescued the impaired endocytosis and EPSC attenuation induced by intraterminal WT h-tau. Thus, MTs overassembled by soluble WT h-tau protein may capture free dynamin at presynaptic terminals, thereby inhibiting SV endocytosis and synaptic transmission, at least in this slice model. This mechanism of dynamin sequestration by newly assembled MTs may also underlie the toxic effects of α-synuclein on SV endocytosis in PD (46).

[0152] Unlike WT h-tau, FTDP-linked mutant tau does not affect SV endocytosis (32), but it binds to both actin filaments (70) and the SV transmembrane protein synaptogyrin (31), thereby immobilizing SVs (31, 32). WT tau can also bind synaptogyrin (31), but cannot bind F-actin due to differences in the MT-binding domain between FTDP mutant and WT tau (62, 71). However, WT h-tau can bind to various other macromolecules and organelles (e.g., MTs, neurofilaments, and ribosomes) (72), as well as synaptogyrin, potentially immobilizing SVs. SV recycling by this mechanism may further contribute to the attenuation of synaptic transmission that remains uninhibited by MT-dynamin inhibitor peptides. In the absence of powerful tools to alleviate symptoms associated with AD or PD, the Held calyx slice model may provide a platform from which therapeutic tools can be explored to rescue synaptic dysfunction. The combination of this slice model with an animal model may provide a new avenue towards rescuing neurological disorders.

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Claims

1. A preventive and / or therapeutic agent for Alzheimer's disease, comprising a peptide corresponding to dynamin 1.

2. 2. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1, wherein the peptide corresponds to a dynamin 1-pleckstrin homology domain or a dynamin 1-proline-rich domain.

3. 2. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, an amino acid sequence having one or more conservative amino acid substitutions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, or an amino acid sequence having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9.

4. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1 , wherein the peptide is encapsulated in nanoparticles to improve delivery of the peptide to the brain.

5. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1 , wherein the peptide is linked to a peptide sequence that improves delivery of the peptide to the brain.

6. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1, wherein the peptide sequence that improves delivery of the peptide to the brain is selected from the group consisting of SEQ ID NOs: 10 to 15.

7. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 1 , wherein the peptide is fused or conjugated to a compound that improves delivery of the peptide to the brain.

8. A preventive and / or therapeutic agent for Alzheimer's disease, comprising an inhibitor of microtubule-dynamin 1 binding.

9. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 8, wherein the inhibitor is an isolated monoclonal antibody that binds to dynamin 1.

10. 9. The preventive and / or therapeutic agent for Alzheimer's disease according to claim 8, wherein the inhibitor is an isolated monoclonal antibody that binds to the dynamin 1-pleckstrin homology domain or the dynamin 1-proline-rich domain.

11. An isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding, the monoclonal antibody being directed against dynamin 1.

12. 12. The isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding according to claim 11, wherein the monoclonal antibody is an antibody against the dynamin 1-pleckstrin homology domain or the dynamin 1-proline-rich domain.

13. An isolated monoclonal antibody that inhibits microtubule-dynamin 1 binding as described in claim 11, wherein the monoclonal antibody is an antibody against a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, an amino acid sequence having one or more conservative amino acid substitutions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, or an amino acid sequence having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9.

14. A method for screening for a substance effective in treating Alzheimer's disease, comprising measuring the activity of a test substance that inhibits the binding of microtubules and dynamin 1.

Images